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large - scale, multisite observational research studies and pragmatic clinical trials utilize clinical data, including diagnosis data that are encoded with the international classification of diseases, 9th revision, clinical modification (icd-9-cm), collected by health systems as a byproduct of patient care. the national mandate for health systems to migrate to icd-10-cm in october 2015 has an impact on all research activities that rely on these codes. further, many current and ongoing investigations will need to manage and analyze data sets that define conditions of interest (i.e., clinical phenotypes) using both icd-9-cm and icd-10-cm codes. the growing availability of electronic health record (ehr) data (encoded in icd-10-cm) will increasingly be leveraged to support pragmatic clinical trials and quality improvement studies that learning health care comprises. longitudinal studies in progress and retrospective studies will use icd-9-cm - based population definitions and will need to understand how those relate to definitions based upon icd-10-cm. a common challenge for researchers and health administrators moving forward, then, is the need to translate icd-9-cm - based phenotype definitions, which can include hundreds of codes, into icd-10-cm and to ensure that the populations retrieved with those codes are clinically equivalent. although the centers for medicare & medicaid services (cms) has produced general equivalent maps (gems), their use is not straightforward, and different methods for using the gems can result in different outcomes. in the context of pragmatic clinical trials, we explore the use of publicly available mapping files to convert clinical phenotype definitions from icd-9-cm to icd-10-cm, compare the outcome of different approaches, and suggest preferred strategies for using the gems in automated translation. in addition to the phenotype definitions, we also make use of the value sets defined for electronic quality measurement as an additional way to evaluate the mapping methods. quality measurement value sets are lists of codes from standard terminologies used to identify sub - populations of patients sharing certain demographic and clinical characteristics, as defined by a clinical quality measure. these value sets are very similar in their function to phenotype definitions. as part of the cms meaningful use of ehr program, certified systems have to demonstrate the electronic submission of data for some selected clinical quality measures. value sets are published to allow automatic computation of the numerator and denominator of a quality measure. the tremendous costs associated with traditional clinical trials limit their use in addressing the majority of clinical questions and treatment decisions that are based upon insufficient evidence.14 further, the limited generalizability of results inherent in clinical trials has stimulated interest in alternative research models, including observational research and pragmatic trials, to support patient - centered outcomes research.5,6 these alternative research models depend upon access to ehr data collected by health systems as part of the patient care process. the hmo research network (hmorn) and other networks have used electronic health care and claims data to advance our understanding of disease.7,8 while electronic claims data have been used in observational research for decades, the growing adoption of ehrs brings the potential to support more sophisticated research activities, such as cohort selection and randomization, to facilitate prospective and interventional research studies.9,10 the routine use of ehr data is a vital component of the envisaged learning health care system, and has become feasible with the widespread adoption and meaningful use of ehrs in health care systems.11 pragmatic trials are those conducted in actual patient care settings and in cooperation with health care systems.6 the national institutes of health (nih) health care systems research collaboratory is funded by the nih common fund to strengthen the national capacity for implementing cost - effective, large - scale research studies that engage health care delivery organizations as research partners, with the assumption that this will make research results more relevant to providers and, ultimately, patients.12 the collaboratory includes a number of pragmatic trial demonstration projects that are multisite, often cluster - randomized, intervention studies.13 these demonstration projects have developed explicit and reproducible definitions (i.e., clinical phenotypes) using icd-9-cm and other standardized code systems to identify patients with precise clinical attributes from various organizations and heterogeneous ehrs. these clinical phenotype definitions support a number of research activities, including cohort identification and describing the baseline characteristics (e.g., the proportion of patients with diabetes or hypertension) of different patient populations. the phenotype definitions of the nih collaboratory projects currently include codes from icd-9-cm, but investigators need to adapt them to icd-10-cm since health care systems transitioned to it by october 1, 2015. rather, it is a radical transformation, involving major changes in not only the size of the terminology, but also in the organization, granularity, and semantics (or meaning) of terms.14 the more than 68,000 possible terms in icd-10-cm more than quadruple the 14,000 terms in icd-9-cm. because the collaboratory demonstration projects are all multiyear studies that span this national icd-10-cm transition period, investigators need to address both icd-9-cm and icd-10-cm in their research data sets. to ease the burden of researchers who need to translate their cohort or clinical phenotype definitions from icd-9-cm to icd-10-cm, we explored the use of published maps between icd-9-cm and icd-10-cm for automatic conversion. the general equivalent maps (gems) are created and maintained by the centers for medicare & medicaid services (cms) and the centers for disease control and prevention (cdc), and serve as a tool for the conversion of data between icd-9-cm and icd-10-cm.15 the gems are often also referred to as crosswalks since they provide important information linking codes from one system with codes in the other system.16 users are cautioned against using the gems for actual coding as they have not been completely validated for clinical use. however, the conversion of data for quality measures and research is specifically listed among the applicable use cases.16 the gems are directional and therefore have two types : the forward maps convert icd-9-cm codes into icd-10-cm, and the backward maps convert icd-10-cm codes into icd-9-cm. because the relationships between icd-9-cm and icd-10-cm codes are often complex and not one - to - one, the use of gems is complicated and requires informed consideration.1720 while the impact of icd-10-cm transition has been explored in various health care and research settings,2124 there are few studies on the evaluation of automated translation of codes between icd-9-cm and icd-10-cm in the context of phenotype definitions for pragmatic trials. the forward and backward gems are not simple mirror images of each other, as the names may suggest. they are independent maps that differ significantly in scope and coverage (table 1). the majority of icd-10-cm codes are not represented in the forward map, and a significant portion of icd-9-cm codes (25 percent) are not represented in the backward map. the backward map provides 78,034 unique pairs of icd-9-cm and icd-10-cm codes (over three times more than the forward map), of which only 18,484 pairs (23.7 percent) are also found in the forward map. users of the gems often find that they need to apply the forward and backward maps iteratively in order to obtain code maps (or links) that would otherwise be missed. according to boyd.,25 36 percent of the icd-9-cm codes are involved in so - called convoluted mappings, meaning that they are not simple one - to - one, one - to - many, or many - to - one maps to icd-10-cm codes. in these complex cases, iterative application of the forward and backward maps will discover more and more links from an icd-9-cm source code to icd-10-cm targets (see methods section). as an example, consider the icd-9-cm code 648.82 abnormal glucose tolerance of mother, delivered, with mention of postpartum complication. using either the forward or the backward gem alone, one will find the target icd-10-cm code o99.815 abnormal glucose complicating the puerperium. with the iterative use of the two gems, three additional relevant icd-10-cm target codes can be found : o24.430 gestational diabetes mellitus in the puerperium, diet controlledo24.434 gestational diabetes mellitus in the puerperium, insulin controlledo24.439 gestational diabetes mellitus in the puerperium, unspecified control. o24.430 gestational diabetes mellitus in the puerperium, diet controlled o24.434 gestational diabetes mellitus in the puerperium, insulin controlled o24.439 gestational diabetes mellitus in the puerperium, unspecified control. first, it may take many iterations to exhaust all mapping relationships because some of the convoluted mappings are open - ended. second, some of the additional codes discovered in this way are not relevant. the aim of this study is to determine the optimal way to use the gems in the context of icd-9-cm code translation in phenotypic definition. the tremendous costs associated with traditional clinical trials limit their use in addressing the majority of clinical questions and treatment decisions that are based upon insufficient evidence.14 further, the limited generalizability of results inherent in clinical trials has stimulated interest in alternative research models, including observational research and pragmatic trials, to support patient - centered outcomes research.5,6 these alternative research models depend upon access to ehr data collected by health systems as part of the patient care process. the hmo research network (hmorn) and other networks have used electronic health care and claims data to advance our understanding of disease.7,8 while electronic claims data have been used in observational research for decades, the growing adoption of ehrs brings the potential to support more sophisticated research activities, such as cohort selection and randomization, to facilitate prospective and interventional research studies.9,10 the routine use of ehr data is a vital component of the envisaged learning health care system, and has become feasible with the widespread adoption and meaningful use of ehrs in health care systems.11 pragmatic trials are those conducted in actual patient care settings and in cooperation with health care systems.6 the national institutes of health (nih) health care systems research collaboratory is funded by the nih common fund to strengthen the national capacity for implementing cost - effective, large - scale research studies that engage health care delivery organizations as research partners, with the assumption that this will make research results more relevant to providers and, ultimately, patients.12 the collaboratory includes a number of pragmatic trial demonstration projects that are multisite, often cluster - randomized, intervention studies.13 these demonstration projects have developed explicit and reproducible definitions (i.e., clinical phenotypes) using icd-9-cm and other standardized code systems to identify patients with precise clinical attributes from various organizations and heterogeneous ehrs. these clinical phenotype definitions support a number of research activities, including cohort identification and describing the baseline characteristics (e.g., the proportion of patients with diabetes or hypertension) of different patient populations. the phenotype definitions of the nih collaboratory projects currently include codes from icd-9-cm, but investigators need to adapt them to icd-10-cm since health care systems transitioned to it by october 1, 2015. rather, it is a radical transformation, involving major changes in not only the size of the terminology, but also in the organization, granularity, and semantics (or meaning) of terms.14 the more than 68,000 possible terms in icd-10-cm more than quadruple the 14,000 terms in icd-9-cm. because the collaboratory demonstration projects are all multiyear studies that span this national icd-10-cm transition period, investigators need to address both icd-9-cm and icd-10-cm in their research data sets. to ease the burden of researchers who need to translate their cohort or clinical phenotype definitions from icd-9-cm to icd-10-cm, we explored the use of published maps between icd-9-cm and icd-10-cm for automatic conversion. the general equivalent maps (gems) are created and maintained by the centers for medicare & medicaid services (cms) and the centers for disease control and prevention (cdc), and serve as a tool for the conversion of data between icd-9-cm and icd-10-cm.15 the gems are often also referred to as crosswalks since they provide important information linking codes from one system with codes in the other system.16 users are cautioned against using the gems for actual coding as they have not been completely validated for clinical use. however, the conversion of data for quality measures and research is specifically listed among the applicable use cases.16 the gems are directional and therefore have two types : the forward maps convert icd-9-cm codes into icd-10-cm, and the backward maps convert icd-10-cm codes into icd-9-cm. because the relationships between icd-9-cm and icd-10-cm codes are often complex and not one - to - one, the use of gems is complicated and requires informed consideration.1720 while the impact of icd-10-cm transition has been explored in various health care and research settings,2124 there are few studies on the evaluation of automated translation of codes between icd-9-cm and icd-10-cm in the context of phenotype definitions for pragmatic trials. the forward and backward gems are not simple mirror images of each other, as the names may suggest. they are independent maps that differ significantly in scope and coverage (table 1). the majority of icd-10-cm codes are not represented in the forward map, and a significant portion of icd-9-cm codes (25 percent) are not represented in the backward map. the backward map provides 78,034 unique pairs of icd-9-cm and icd-10-cm codes (over three times more than the forward map), of which only 18,484 pairs (23.7 percent) are also found in the forward map. users of the gems often find that they need to apply the forward and backward maps iteratively in order to obtain code maps (or links) that would otherwise be missed. according to boyd.,25 36 percent of the icd-9-cm codes are involved in so - called convoluted mappings, meaning that they are not simple one - to - one, one - to - many, or many - to - one maps to icd-10-cm codes. in these complex cases, iterative application of the forward and backward maps will discover more and more links from an icd-9-cm source code to icd-10-cm targets (see methods section). as an example, consider the icd-9-cm code 648.82 abnormal glucose tolerance of mother, delivered, with mention of postpartum complication. using either the forward or the backward gem alone, one will find the target icd-10-cm code o99.815 abnormal glucose complicating the puerperium. with the iterative use of the two gems, three additional relevant icd-10-cm target codes can be found : o24.430 gestational diabetes mellitus in the puerperium, diet controlledo24.434 gestational diabetes mellitus in the puerperium, insulin controlledo24.439 gestational diabetes mellitus in the puerperium, unspecified control. o24.430 gestational diabetes mellitus in the puerperium, diet controlled o24.434 gestational diabetes mellitus in the puerperium, insulin controlled o24.439 gestational diabetes mellitus in the puerperium, unspecified control. first, it may take many iterations to exhaust all mapping relationships because some of the convoluted mappings are open - ended. the aim of this study is to determine the optimal way to use the gems in the context of icd-9-cm code translation in phenotypic definition. in this study, we compared four progressively more aggressive methods for using the gems (figure 1). the goal of each method was to identify, for each icd-9-cm code (the source code), one or more corresponding icd-10-cm codes (the target codes). for all methods, we used a combination of the forward and backward gems to discover linkages between icd-9-cm and icd-10-cm codes. we treated the linkages in the forward and backward gems as the same, ignoring the stated directionality of the maps. in increasing order of aggressiveness, the methods are the following : simple forward map (sfm) : all icd-10-cm codes linked to an icd-9-cm code in the forward gem are used as targets.forward backward map (fbm) : uses direct links from both the forward and backward gems. this includes all maps in sfm, plus additional map targets identified by the links between icd-9-cm and icd-10-cm codes in the backward gem.secondary map (sm) : uses all maps in fbm, plus additional target codes identified by secondary icd-9-cm codes.the following are the steps to generate sm : based on fbm, identify secondary icd-9-cm codes, which are defined as icd-9-cm codes that share the same target icd-10-cm code as the primary icd-9-cm source code. in figure 1, consider the (primary) icd-9-cm source code a. it has targets w and x in fbm ; while another icd-9-cm code b has targets x and y in fbm. since a and b share the same target x, b is identified as a secondary code of a.add the targets of the secondary codes in fbm to the list of targets for the primary source code. in figure 1, x and y are added as targets for source code a.tertiary map (tm) : uses all maps in sm, plus additional target codes identified by tertiary icd-9-cm codes.the following are the steps to generate tm : based on fbm, identify tertiary icd-9-cm codes, which are defined as icd-9-cm codes that share the same icd-10-cm code as the secondary icd-9-cm code (identified in the generation of sm). in figure 1, b has been identified as a secondary code to primary source code a. in fbm, b has targets x and y, while c has targets y and z. since b and c share the same target y, c is identified as a tertiary code of a.add the targets of the tertiary codes in fbm to the list of targets for the primary source code. in figure 1, y and z are added as targets for source code a. simple forward map (sfm) : uses only direct links from the forward gem. all icd-10-cm codes linked to an icd-9-cm code in the forward gem are used as targets. forward backward map (fbm) : uses direct links from both the forward and backward gems. this includes all maps in sfm, plus additional map targets identified by the links between icd-9-cm and icd-10-cm codes in the backward gem. secondary map (sm) : uses all maps in fbm, plus additional target codes identified by secondary icd-9-cm codes. the following are the steps to generate sm : based on fbm, identify secondary icd-9-cm codes, which are defined as icd-9-cm codes that share the same target icd-10-cm code as the primary icd-9-cm source code. in figure 1, consider the (primary) icd-9-cm source code a. it has targets w and x in fbm ; while another icd-9-cm code b has targets x and y in fbm. since a and b share the same target x, b is identified as a secondary code of a.add the targets of the secondary codes in fbm to the list of targets for the primary source code. in figure 1, x and y are added as targets for source code a. based on fbm, identify secondary icd-9-cm codes, which are defined as icd-9-cm codes that share the same target icd-10-cm code as the primary icd-9-cm source code. in figure 1, consider the (primary) icd-9-cm source code a. it has targets w and x in fbm ; while another icd-9-cm code b has targets x and y in fbm. since a and b share the same target x, b is identified as a secondary code of a. add the targets of the secondary codes in fbm to the list of targets for the primary source code. in figure 1, x and y are added as targets for source code a. tertiary map (tm) : uses all maps in sm, plus additional target codes identified by tertiary icd-9-cm codes. the following are the steps to generate tm : based on fbm, identify tertiary icd-9-cm codes, which are defined as icd-9-cm codes that share the same icd-10-cm code as the secondary icd-9-cm code (identified in the generation of sm). in figure 1, b has been identified as a secondary code to primary source code a. in fbm, b has targets x and y, while c has targets y and z. since b and c share the same target y, c is identified as a tertiary code of a.add the targets of the tertiary codes in fbm to the list of targets for the primary source code. in figure 1, y and z are added as targets for source code a. based on fbm, identify tertiary icd-9-cm codes, which are defined as icd-9-cm codes that share the same icd-10-cm code as the secondary icd-9-cm code (identified in the generation of sm). in figure 1, b has been identified as a secondary code to primary source code a. in fbm, b has targets x and y, while c has targets y and z. since b and c share the same target y, c is identified as a tertiary code of a. add the targets of the tertiary codes in fbm to the list of targets for the primary source code. in figure 1, y and z are added as targets for source code a. we chose these four methods for a number of reasons. the sfm and fbm are the most common ways to use the gems, and will provide a baseline measure of mapping performance. sm corresponds to the method used in the online transition tool provided by boyd s group.26 given their experience and commentary, we hypothesized that additional iterations will increase the number of icd-10-cm target codes and may enhance mapping performance. therefore tm was included to assess whether additional iterations are indeed beneficial. to evaluate the performance of the four mapping methods, we used a convenience sample of 32 phenotypes (developed to identify research cohorts, characterize risk factors, or define outcomes) from three different pragmatic trials collaborative care for chronic pain in primary care (ppact), strategies and opportunities to stop colorectal cancer in priority populations (stop crc), and a pragmatic trial of population - based programs to prevent suicide attempt that were defined by icd-9-cm codes. the icd-9-cm codes were translated to icd-10-cm codes using the four mapping methods based on the 2014 version of the gems. one generalist nurse practitioner (kp) and an md domain expert for each trial (bg, ap, and mc) reviewed the phenotype name and the icd-10-cm code sets generated by the maps to determine if each icd-10-cm code semantically fit into the named phenotype condition, based on their understanding of that phenotype and its intent. for example, for the phenotype active alcohol abuse the reviewer was asked to look at the icd-10-cm codes and determine (yes or no) if those codes were appropriate for inclusion in that heading. reviewers were provided the original phenotype definition (i.e., the set of icd-9-cm codes that constitute the specified condition) as a reference on the same review sheet. to limit the scope and time for the evaluation, the reviewers were asked to review only the icd-10-cm codes generated by the different mapping methods. they were not asked to search for additional icd-10-cm codes that should have been included. to shorten the list of icd-10-cm codes for review, we derived an algorithm to roll up codes to their parents, as long as the total number of codes in the list was reduced. for example, if the list contained m47.10, m47.11, m47.12, m47.13, m47.15, m47.16, which were all children of m47.1, we converted it into m47.1 exclusion : m47.14 because m47.14 was the only child of m47.1 not included in the list. we did this iteratively until no further reduction in the number of codes was possible. to evaluate the roll up algorithm, one cohort definition from each demonstration project with at least 10 icd-10-cm codes was manually reviewed to make sure that the final list of codes represented the meaning of the original codes. to obtain the quality measurement value sets, we used the value set authority center (vsac) launched by the united states national library of medicine (nlm) in 2012 to provide access to all official versions of value sets.27,28 in the vsac, we identified all value sets for 2014 clinical quality measures that were dually defined with both icd-9-cm and icd-10-cm code lists. we applied the four mapping methods to the icd-9-cm code lists, and evaluated the resulting icd-10-cm target codes against the icd-10-cm codes listed for that measure, using the latter as the gold standard. since the value sets differed considerably in their sizes, we also analyzed the effect of value set size on the mapping performance. to evaluate the performance of each mapping method, we calculated the recall, precision and f - score of each method for every phenotype definition and quality measure value set. note that for the phenotype definitions, we did not measure the true recall because the reviewers were not asked to look for missing icd-10-cm codes. to give an estimate of recall for the phenotype definitions, we assumed that the most aggressive method (tm) contained all the correct icd-10-cm codes. we used the f - score (the harmonic mean between recall and precision) as an overall indicator of performance of each mapping method. based on the distribution of the f - scores, we used the anova test to check the statistical significance of the difference between the four methods. in this study, we compared four progressively more aggressive methods for using the gems (figure 1). the goal of each method was to identify, for each icd-9-cm code (the source code), one or more corresponding icd-10-cm codes (the target codes). for all methods, we used a combination of the forward and backward gems to discover linkages between icd-9-cm and icd-10-cm codes. we treated the linkages in the forward and backward gems as the same, ignoring the stated directionality of the maps. in increasing order of aggressiveness, the methods are the following : simple forward map (sfm) : all icd-10-cm codes linked to an icd-9-cm code in the forward gem are used as targets.forward backward map (fbm) : uses direct links from both the forward and backward gems. this includes all maps in sfm, plus additional map targets identified by the links between icd-9-cm and icd-10-cm codes in the backward gem.secondary map (sm) : uses all maps in fbm, plus additional target codes identified by secondary icd-9-cm codes.the following are the steps to generate sm : based on fbm, identify secondary icd-9-cm codes, which are defined as icd-9-cm codes that share the same target icd-10-cm code as the primary icd-9-cm source code. in figure 1, consider the (primary) icd-9-cm source code a. it has targets w and x in fbm ; while another icd-9-cm code b has targets x and y in fbm. since a and b share the same target x, b is identified as a secondary code of a.add the targets of the secondary codes in fbm to the list of targets for the primary source code. in figure 1, x and y are added as targets for source code a.tertiary map (tm) : uses all maps in sm, plus additional target codes identified by tertiary icd-9-cm codes.the following are the steps to generate tm : based on fbm, identify tertiary icd-9-cm codes, which are defined as icd-9-cm codes that share the same icd-10-cm code as the secondary icd-9-cm code (identified in the generation of sm). in figure 1, b has been identified as a secondary code to primary source code a. in fbm, b has targets x and y, while c has targets y and z. since b and c share the same target y, c is identified as a tertiary code of a.add the targets of the tertiary codes in fbm to the list of targets for the primary source code. in figure 1, y and z are added as targets for source code a. simple forward map (sfm) : uses only direct links from the forward gem. all icd-10-cm codes linked to an icd-9-cm code in the forward gem are used as targets. forward backward map (fbm) : uses direct links from both the forward and backward gems. this includes all maps in sfm, plus additional map targets identified by the links between icd-9-cm and icd-10-cm codes in the backward gem. secondary map (sm) : uses all maps in fbm, plus additional target codes identified by secondary icd-9-cm codes. the following are the steps to generate sm : based on fbm, identify secondary icd-9-cm codes, which are defined as icd-9-cm codes that share the same target icd-10-cm code as the primary icd-9-cm source code. in figure 1, consider the (primary) icd-9-cm source code a. it has targets w and x in fbm ; while another icd-9-cm code b has targets x and y in fbm. since a and b share the same target x, b is identified as a secondary code of a.add the targets of the secondary codes in fbm to the list of targets for the primary source code. in figure 1, x and y are added as targets for source code a. based on fbm, identify secondary icd-9-cm codes, which are defined as icd-9-cm codes that share the same target icd-10-cm code as the primary icd-9-cm source code. in figure 1, consider the (primary) icd-9-cm source code a. it has targets w and x in fbm ; while another icd-9-cm code b has targets x and y in fbm. since a and b share the same target x, b is identified as a secondary code of a. add the targets of the secondary codes in fbm to the list of targets for the primary source code. in figure 1, x and y are added as targets for source code a. tertiary map (tm) : uses all maps in sm, plus additional target codes identified by tertiary icd-9-cm codes. the following are the steps to generate tm : based on fbm, identify tertiary icd-9-cm codes, which are defined as icd-9-cm codes that share the same icd-10-cm code as the secondary icd-9-cm code (identified in the generation of sm). in figure 1, b has been identified as a secondary code to primary source code a. in fbm, b has targets x and y, while c has targets y and z. since b and c share the same target y, c is identified as a tertiary code of a.add the targets of the tertiary codes in fbm to the list of targets for the primary source code. in figure 1, y and z are added as targets for source code a. based on fbm, identify tertiary icd-9-cm codes, which are defined as icd-9-cm codes that share the same icd-10-cm code as the secondary icd-9-cm code (identified in the generation of sm). in figure 1, b has been identified as a secondary code to primary source code a. in fbm, b has targets x and y, while c has targets y and z. since b and c share the same target y, c is identified as a tertiary code of a. add the targets of the tertiary codes in fbm to the list of targets for the primary source code. in figure 1, y and z are added as targets for source code a. we chose these four methods for a number of reasons. the sfm and fbm are the most common ways to use the gems, and will provide a baseline measure of mapping performance. sm corresponds to the method used in the online transition tool provided by boyd s group.26 given their experience and commentary, we hypothesized that additional iterations will increase the number of icd-10-cm target codes and may enhance mapping performance. to evaluate the performance of the four mapping methods, we used a convenience sample of 32 phenotypes (developed to identify research cohorts, characterize risk factors, or define outcomes) from three different pragmatic trials collaborative care for chronic pain in primary care (ppact), strategies and opportunities to stop colorectal cancer in priority populations (stop crc), and a pragmatic trial of population - based programs to prevent suicide attempt that were defined by icd-9-cm codes. the icd-9-cm codes were translated to icd-10-cm codes using the four mapping methods based on the 2014 version of the gems. one generalist nurse practitioner (kp) and an md domain expert for each trial (bg, ap, and mc) reviewed the phenotype name and the icd-10-cm code sets generated by the maps to determine if each icd-10-cm code semantically fit into the named phenotype condition, based on their understanding of that phenotype and its intent. for example, for the phenotype active alcohol abuse the reviewer was asked to look at the icd-10-cm codes and determine (yes or no) if those codes were appropriate for inclusion in that heading. reviewers were provided the original phenotype definition (i.e., the set of icd-9-cm codes that constitute the specified condition) as a reference on the same review sheet. to limit the scope and time for the evaluation, the reviewers were asked to review only the icd-10-cm codes generated by the different mapping methods. they were not asked to search for additional icd-10-cm codes that should have been included. to shorten the list of icd-10-cm codes for review codes to their parents, as long as the total number of codes in the list was reduced. m47.10, m47.11, m47.12, m47.13, m47.15, m47.16, which were all children of m47.1, we converted it into m47.1 exclusion : m47.14 because m47.14 was the only child of m47.1 not included in the list. we did this iteratively until no further reduction in the number of codes was possible. to evaluate the roll up algorithm, one cohort definition from each demonstration project with at least 10 icd-10-cm codes was manually reviewed to make sure that the final list of codes represented the meaning of the original codes. to obtain the quality measurement value sets, we used the value set authority center (vsac) launched by the united states national library of medicine (nlm) in 2012 to provide access to all official versions of value sets.27,28 in the vsac, we identified all value sets for 2014 clinical quality measures that were dually defined with both icd-9-cm and icd-10-cm code lists. we applied the four mapping methods to the icd-9-cm code lists, and evaluated the resulting icd-10-cm target codes against the icd-10-cm codes listed for that measure, using the latter as the gold standard. since the value sets differed considerably in their sizes, we also analyzed the effect of value set size on the mapping performance. to evaluate the performance of each mapping method, we calculated the recall, precision and f - score of each method for every phenotype definition and quality measure value set. note that for the phenotype definitions, we did not measure the true recall because the reviewers were not asked to look for missing icd-10-cm codes. to give an estimate of recall for the phenotype definitions, we assumed that the most aggressive method (tm) contained all the correct icd-10-cm codes. we used the f - score (the harmonic mean between recall and precision) as an overall indicator of performance of each mapping method. based on the distribution of the f - scores, we used the anova test to check the statistical significance of the difference between the four methods. the selected pragmatic trials used 32 cohort definitions with 3161 (median 4) icd-9-cm codes per definition (table 2). there were altogether 536 unique icd-9-cm codes, all of which could be mapped by the four different methods. the size of the resulting icd-10-cm code sets progressively increased as more aggressive mapping methods were used. overall for sfm, the median size of the icd-10-cm code sets was comparable to their icd-9-cm counterparts. there was a sharp increase from sfm to fbm, and also from fbm to sm. our roll up algorithm reduced the review workload to around 2,000 codes. to ensure that the shortened list of codes represented the same meaning as the original codes, we reviewed three cohort definitions (pelvic and abdominal pain, alcohol abuse, and colon cancer) with 14, 40, and 80 leaf level icd-10-cm codes respectively. our roll up algorithm collapsed the lists to 5 codes (1 higher level code, 4 leaf codes), 11 codes (4 high level codes, 2 leaf codes, 5 exclusion codes) and 20 codes (11 high level codes, 9 leaf codes) respectively. by comparing the meaning of the original codes to the shortened lists, we confirmed that the shortened lists were semantically the same as the original lists. the recall, precision, and f score values are the means for the code sets in a demonstration project. fbm was better than sfm in all three metrics (precision, recall, and f score). as expected, the more aggressive methods sm and tm resulted in higher recall at the expense of precision. using the overall mean f score as a single indicator of performance, fbm was the best (f=0.67), but was close to sm (f=0.62) and tm (f=0.60). based on the distribution of individual f scores in each method, the overall difference between the four methods was statistically significant (one - way anova, f=5.749, p=0.001). pairwise comparison between adjacent pairs of methods by paired samples t - test showed that the difference between sfm and fbm was statistically significant (t=6.184, p<0.0001), while the differences for fbm versus sm and sm versus tm were not. a total of 202 quality measure value sets defined by both icd-9-cm and icd-10-cm code sets were retrieved from the vsac. there were altogether 5,545 unique icd-9-cm codes, of which 2 codes could not be mapped by our selected methods because they were not included in either the forward or backward gem. the performance of the mapping methods in relation to the size of the icd-9-cm code sets is summarized in table 4. the recall, precision, and f - score values shown are the means for the value sets within a particular size range. based on the overall f - score, the overall best performing mapping method was fbm, followed by sm, tm, and sfm. based on the distribution of f - scores for each value set, the difference in the performance of the four methods was statistically significant (one - way anova, f=40.889, p<0.0005). pairwise comparisons between adjacent methods (sfm versus fbm, fbm versus sm and sm versus tm) by paired samples t - test were all statistically significant (all with p<0.0001). the number of icd-9-cm codes in the value sets varied considerably from 1 to 1,212 (mean 58.6, median 6). smaller value sets generally had better recall, precision, and f - scores, regardless of mapping method. for fbm, value sets with 20 or fewer codes had almost perfect recall (0.97) and precision (0.93). the selected pragmatic trials used 32 cohort definitions with 3161 (median 4) icd-9-cm codes per definition (table 2). there were altogether 536 unique icd-9-cm codes, all of which could be mapped by the four different methods. the size of the resulting icd-10-cm code sets progressively increased as more aggressive mapping methods were used. overall for sfm, the median size of the icd-10-cm code sets was comparable to their icd-9-cm counterparts. there was a sharp increase from sfm to fbm, and also from fbm to sm. our roll up algorithm reduced the review workload to around 2,000 codes. to ensure that the shortened list of codes represented the same meaning as the original codes, we reviewed three cohort definitions (pelvic and abdominal pain, alcohol abuse, and colon cancer) with 14, 40, and 80 leaf level icd-10-cm codes respectively. our roll up algorithm collapsed the lists to 5 codes (1 higher level code, 4 leaf codes), 11 codes (4 high level codes, 2 leaf codes, 5 exclusion codes) and 20 codes (11 high level codes, 9 leaf codes) respectively. by comparing the meaning of the original codes to the shortened lists, we confirmed that the shortened lists were semantically the same as the original lists. the recall, precision, and f score values are the means for the code sets in a demonstration project. fbm was better than sfm in all three metrics (precision, recall, and f score). as expected, the more aggressive methods sm and tm resulted in higher recall at the expense of precision. using the overall mean f score as a single indicator of performance, fbm was the best (f=0.67), but was close to sm (f=0.62) and tm (f=0.60). based on the distribution of individual f scores in each method, the overall difference between the four methods was statistically significant (one - way anova, f=5.749, p=0.001). pairwise comparison between adjacent pairs of methods by paired samples t - test showed that the difference between sfm and fbm was statistically significant (t=6.184, p<0.0001), while the differences for fbm versus sm and sm versus tm were not. a total of 202 quality measure value sets defined by both icd-9-cm and icd-10-cm code sets were retrieved from the vsac. there were altogether 5,545 unique icd-9-cm codes, of which 2 codes could not be mapped by our selected methods because they were not included in either the forward or backward gem. the performance of the mapping methods in relation to the size of the icd-9-cm code sets is summarized in table 4. the recall, precision, and f - score values shown are the means for the value sets within a particular size range. based on the overall f - score, the overall best performing mapping method was fbm, followed by sm, tm, and sfm. based on the distribution of f - scores for each value set, the difference in the performance of the four methods was statistically significant (one - way anova, f=40.889, p<0.0005). pairwise comparisons between adjacent methods (sfm versus fbm, fbm versus sm and sm versus tm) by paired samples t - test were all statistically significant (all with p<0.0001). the number of icd-9-cm codes in the value sets varied considerably from 1 to 1,212 (mean 58.6, median 6). smaller value sets generally had better recall, precision, and f - scores, regardless of mapping method. for fbm, value sets with 20 or fewer codes had almost perfect recall (0.97) and precision (0.93). after several false starts and delays, the transition from icd-9-cm to icd-10-cm finally happened in 2015. health care providers have adopted the new coding system to ensure continued revenue ; researchers and other secondary users of health care data must be prepared to adapt to this change. after october 1, 2015, phenotype definitions that use icd-9-cm codes to identify cohorts of patients have to shift to icd-10-cm codes if they are applied to newly - collected data. these icd-9-cm - based phenotype definitions can include hundreds of codes. translating them into icd-10-cm entails significant effort, and automated methods to support these translations reduce this burden. the use of the gems is not straightforward because it includes two independent maps in both directions. different methods for using the gems will result in different outcomes, and our findings can inform optimal approaches to using the maps for automated translation. in this study, we compare four progressively aggressive methods of using the gems to translate icd-9-cm codes used in phenotype definitions to icd-10-cm codes : (1) simple forward map (sfm) ; (2) forward backward map (fbm) ; (3) secondary map (sm) ; and (4) tertiary map (tm). the papers and online tool from boyd. seem to favor an approach similar to sm, but they did not explain why, nor did they compare the various mapping methods quantitatively.21,25,29 in our study, the different methods are compared quantitatively, and their strengths and weakness are highlighted. the poor results from the simple forward map should caution novice users of the gems, who may believe that using the forward map alone will be sufficient to translate icd-9-cm codes to icd-10-cm. since the majority of icd-10-cm codes (75 percent) are not reachable by the forward map, it is not surprising that the performance of sfm is the worst. the two gems together include 13,478 (93 percent) of icd-9-cm codes and 69,154 (99 percent) of icd-10-cm codes. this is an absolute limitation for any mapping method relying on the gems alone, which means that there is a small percentage of icd-9-cm (7 percent) and icd-10-cm (1 percent) codes that will not be covered. boyd. demonstrated that the majority of the icd-9-cm to icd-10-cm translations are complex, convoluted, and nonreciprocal.29 this is why one needs to apply the forward and backward maps iteratively to obtain more complete results. in our study, sm (the first iteration) identified several times more icd-10-cm codes than did fbm. a common source of error related to composite concepts involving more than one medical condition. for example, starting from the icd-9-cm code 716.80 other specified arthropathy, site unspecified the fbm found e08.618 diabetes mellitus due to underlying condition with other diabetic arthropathy, which was a correct target. however, in the sm, e08.618 led to the identification of the secondary icd-9-cm code 249.80 secondary diabetes mellitus with other specified manifestations, not stated as uncontrolled, or unspecified. this secondary icd-9-cm code led to additional icd-10-cm targets, such as e10.621 type 1 diabetes mellitus with foot ulcer, which were completely unrelated to the primary icd-9-cm source code. such examples highlight the need for thoughtfulness and manual review of mappings generated by aggressive iterative mapping methods. based on the f - scores, the fbm was the best performing among all methods (the complete fbm list is available as appendix a). however, the sm was a close second. for the clinical phenotype use case, sm had a better recall (0.89) over fbm (0.68), but precision dropped considerably (from 0.76 to 0.56). the median number of icd-10-cm target codes increased six times from fbm to sm, and only one - third of the additional icd-10-cm codes identified were correct. in practice, the optimal method will depend upon the specific use case, particularly whether higher recall is considered more important than precision or vice versa. in our limited sample of medical conditions, it appears that for clinically distinct and homogenous conditions, such as colorectal cancer, the recall of fbm is already very good, and there is no need to go to more aggressive methods. conditions with heterogeneous pathology involving multiple organ systems, e.g., chronic pain, might require more aggressive mapping methods. in general, the performance of all mapping methods dropped considerably with diverse and heterogeneous conditions. in such cases, manual review of the map - generated codes and search for missing codes will be essential. users of the gems should be aware that only billable codes (leaf codes) are included in the gems. if their code lists include nonbillable (high level codes), such as some code lists used in quality metrics, they should expand the high level codes to the leaf codes before applying the maps to ensure a more complete translation. the performance of gem - based mapping also depends on the extent of the changes between icd-9-cm and icd-10-cm. some chapters such as mental and behavioral disorders and diseases of the skin and subcutaneous tissues have undergone major reorganization. the definitions in the suicide prevention project mostly fall within mental health disorders. as a result, all mapping methods performed more poorly for this group., this condition has four codes depending on whether it is continuous, episodic, in remission, or unspecified. icd-10-cm does not distinguish between the time course (which explains why the sfm maps have less icd-10-cm codes than icd-9-cm codes), but has added a new axis of classification about the effects of the abuse (e.g., sleep disorder, sexual dysfunction). in the chapters that have changed significantly, more intense scrutiny of gem - based translations is warranted, and users may need to look outside the gems for codes that are not reachable through the gems. regardless of the mapping method, our results suggest that automatic translation is not perfect and validation by human review is recommended. however, it is likely that automated translation will save time by reducing the scope of review. the burden of manual review is a real concern, especially in codes sets with hundreds of codes. very often, all descendants of a subbranch are included in a phenotype definition, so it saves significant time for reviewers if codes are rolled up to their parents. with our roll up algorithm, we managed to reduce the number of codes requiring expert review by 72 percent. while clinical quality measurement and pragmatic clinical trials are distinct activities, they both rely on code sets to identify their relevant subpopulations of patients, and there is clear overlap in the function between the phenotype definition code sets and quality measurement value sets. for example, there is a phenotype code set for colon cancer in the nih collaboratory, and a quality measure value set for malignant neoplasm of colon, and both have exactly the same icd-9-cm codes. because of this, we have included the quality measure value sets as an additional evaluation of the mapping methods. for the quality measure value sets, the performance of the four mapping methods followed essentially the same trend as in phenotype code sets. based on the overall f - scores, fbm performed best, followed by sm, tm, and sfm. the more aggressive methods (sm and tm) resulted in only marginal increase in recall with considerable drop in precision. therefore, if there is a need to use the gems to translate icd-9-cm code sets for clinical quality measurement, it would seem appropriate to use the fbm mapping method. note that mapping performance is generally better with smaller value sets. one possible explanation is that smaller value sets may involve more distinct and homogenous conditions (e.g., malignant neoplasm of colon, 13 codes), and that larger value sets tend to be more heterogeneous (e.g., immunocompromised conditions, 149 codes). as we found in cohort definitions, generally, for small value set (fewer than 20 codes) involving homogeneous conditions that are not in the chapters known to have undergone major reorganization in icd-10-cm (e.g., mental disorders), the fbm mappings are expected to perform very well, and only minimal manual review will be required. the existence of code sets used for phenotype definition and quality measurement raises the interesting possibility of cross - fertilization. it is conceivable that, in some cases, the same set of codes can serve both functions, as in the colon cancer example above. indeed, the icd-10-cm codes in the colon cancer value set are all considered appropriate for phenotype definition by the reviewers. so instead of defining new icd-10-cm code sets from scratch, the researchers may be able to find quality value sets defined with icd-10-cm codes that they can reuse. however, to do that one has to search through the thousands of value sets in vsac. to narrow the search, one can use some similarity measure (e.g., jaccard coefficient) between the icd-9-cm phenotype code sets and icd-9-cm value sets in vsac.30 in the future, this kind of cross - fertilization between various secondary uses of clinical codes will become more important and perhaps encourage health care organizations to participate in pragmatic trials and nationally coordinated biomedical and health services research, such as hmorn and the patient centered outcomes research network (pcornet). the phenotype knowledge base (phekb)31 and other repositories of phenotypes should consider partnerships with vsac and investigate formal linkages between research phenotypes and quality measurement value sets. some of the phenotype definitions used in this study are posted on phekb. the use of common value sets for clinical research and quality measurement can enable the generation of evidence from health care organizations and facilitate the vision of learning health care.32,33 for future work, we can explore ways to improve the performance of the mapping methods. there is additional information in the gems, such as flags for approximate or exact maps, and indicators of combination codes, which can be exploited to refine the mapping algorithms. boyd. showed that the mapping relationships for codes from different icd-9-cm chapters varied considerably.28 this is because the difference between icd-9-cm and icd-10-cm is not uniform across all medical specialties. chapters that do not change radically may require a less aggressive mapping approach. outside the use of the gems, two additional mapping resources first, the international health terminology standards development organisation (ihtsdo) publishes a map from systematized nomenclature of medicine clinical terms (snomed ct) to icd-9-cm, and the nlm publishes a map from snomed ct to icd-10-cm. therefore, it is possible to map from icd-9-cm to icd-10-cm using snomed ct as an intermediary. second, the unified medical language system (umls) has been found to be useful in interterminology mapping.34,35 mapping relations between icd-9-cm and icd-10-cm can be discovered by exploring the synonymy and other relationships within the umls. these relationships can then be used to corroborate or supplement the maps derived from the gems. in the future snomed ct is a better clinical terminology than icd because of its coverage, granularity, clinical orientation, and logical underpinning.36 many quality value sets are already defined in snomed ct codes. although it is true that icd codes are more commonly found in ehrs at present, snomed ct codes will become more ubiquitous with the meaningful use initiative. the collaboratory demonstration projects we used were a convenience sample and are not representative of all pragmatic trials. the phenotype definitions in this study were developed to support a number of purposes for very specific research studies and might not be generalizable or appropriate for other research or quality measurement use cases related to those conditions. although we did use two reviewers for each mapping relationship, the reviews by clinical experts have not been independently corroborated. for example, medication or laboratory values can be used to identify more records for potentially complete recall sets. a future related activity would be to examine a known subset of patients with chronic diseases before and after the icd-10-cm transition, and to contrast the assigned icd-10-cm codes with historical icd-9-cm codes. obviously, any practical migration of phenotyping algorithms from icd-9-cm to icd-10-cm will ultimately require the use and synthesis of other data types for validation and human review, including a more rigorously defined characterization of a gold standard diagnosis. even with rigorous gems mapping approaches, real - world application would require some level of human review to consider phenotype definitions fully validated. it is important to mention that although we focused our investigation on the translation of icd - based phenotype definitions, most phenotyping methods include other data, such as laboratory test results, medications, demographics, and natural language processing, in addition to diagnostic code value sets. while most researchers recognize that icd code sets by themselves are not sufficient for research phenotyping, these codes are widely used and remain an important component of virtually all of the phenotype definitions posted on phekb. given the national impact of implementation to icd-10-cm in october 2015, specific scrutiny of public gems tools is warranted to clarify the research implications of the icd-9-cm to icd-10-cm transition. after several false starts and delays, the transition from icd-9-cm to icd-10-cm finally happened in 2015. health care providers have adopted the new coding system to ensure continued revenue ; researchers and other secondary users of health care data must be prepared to adapt to this change. after october 1, 2015, phenotype definitions that use icd-9-cm codes to identify cohorts of patients have to shift to icd-10-cm codes if they are applied to newly - collected data. these icd-9-cm - based phenotype definitions can include hundreds of codes. translating them into icd-10-cm entails significant effort, and automated methods to support these translations reduce this burden. the use of the gems is not straightforward because it includes two independent maps in both directions. different methods for using the gems will result in different outcomes, and our findings can inform optimal approaches to using the maps for automated translation. in this study, we compare four progressively aggressive methods of using the gems to translate icd-9-cm codes used in phenotype definitions to icd-10-cm codes : (1) simple forward map (sfm) ; (2) forward backward map (fbm) ; (3) secondary map (sm) ; and (4) tertiary map (tm). the papers and online tool from boyd. seem to favor an approach similar to sm, but they did not explain why, nor did they compare the various mapping methods quantitatively.21,25,29 in our study, the different methods are compared quantitatively, and their strengths and weakness are highlighted. the poor results from the simple forward map should caution novice users of the gems, who may believe that using the forward map alone will be sufficient to translate icd-9-cm codes to icd-10-cm. since the majority of icd-10-cm codes (75 percent) are not reachable by the forward map, it is not surprising that the performance of sfm is the worst. the two gems together include 13,478 (93 percent) of icd-9-cm codes and 69,154 (99 percent) of icd-10-cm codes. this is an absolute limitation for any mapping method relying on the gems alone, which means that there is a small percentage of icd-9-cm (7 percent) and icd-10-cm (1 percent) codes that will not be covered. boyd. demonstrated that the majority of the icd-9-cm to icd-10-cm translations are complex, convoluted, and nonreciprocal.29 this is why one needs to apply the forward and backward maps iteratively to obtain more complete results. in our study, sm (the first iteration) identified several times more icd-10-cm codes than did fbm. a common source of error related to composite concepts involving more than one medical condition. for example, starting from the icd-9-cm code 716.80 other specified arthropathy, site unspecified the fbm found e08.618 diabetes mellitus due to underlying condition with other diabetic arthropathy, which was a correct target. however, in the sm, e08.618 led to the identification of the secondary icd-9-cm code 249.80 secondary diabetes mellitus with other specified manifestations, not stated as uncontrolled, or unspecified. this secondary icd-9-cm code led to additional icd-10-cm targets, such as e10.621 type 1 diabetes mellitus with foot ulcer, which were completely unrelated to the primary icd-9-cm source code. such examples highlight the need for thoughtfulness and manual review of mappings generated by aggressive iterative mapping methods. based on the f - scores, the fbm was the best performing among all methods (the complete fbm list is available as appendix a). however, the sm was a close second. for the clinical phenotype use case, sm had a better recall (0.89) over fbm (0.68), but precision dropped considerably (from 0.76 to 0.56). the median number of icd-10-cm target codes increased six times from fbm to sm, and only one - third of the additional icd-10-cm codes identified were correct. in practice, the optimal method will depend upon the specific use case, particularly whether higher recall is considered more important than precision or vice versa. in our limited sample of medical conditions, it appears that for clinically distinct and homogenous conditions, such as colorectal cancer, the recall of fbm is already very good, and there is no need to go to more aggressive methods. conditions with heterogeneous pathology involving multiple organ systems, e.g., chronic pain, might require more aggressive mapping methods. in general, the performance of all mapping methods dropped considerably with diverse and heterogeneous conditions. in such cases, manual review of the map - generated codes and search for missing codes will be essential. users of the gems should be aware that only billable codes (leaf codes) are included in the gems. if their code lists include nonbillable (high level codes), such as some code lists used in quality metrics, they should expand the high level codes to the leaf codes before applying the maps to ensure a more complete translation. the performance of gem - based mapping also depends on the extent of the changes between icd-9-cm and icd-10-cm. some chapters such as mental and behavioral disorders and diseases of the skin and subcutaneous tissues have undergone major reorganization. the definitions in the suicide prevention project mostly fall within mental health disorders. as a result, all mapping methods performed more poorly for this group., this condition has four codes depending on whether it is continuous, episodic, in remission, or unspecified. icd-10-cm does not distinguish between the time course (which explains why the sfm maps have less icd-10-cm codes than icd-9-cm codes), but has added a new axis of classification about the effects of the abuse (e.g., sleep disorder, sexual dysfunction). in the chapters that have changed significantly, more intense scrutiny of gem - based translations is warranted, and users may need to look outside the gems for codes that are not reachable through the gems. regardless of the mapping method, our results suggest that automatic translation is not perfect and validation by human review is recommended. however, it is likely that automated translation will save time by reducing the scope of review. the burden of manual review is a real concern, especially in codes sets with hundreds of codes. very often, all descendants of a subbranch are included in a phenotype definition, so it saves significant time for reviewers if codes are rolled up to their parents. with our roll up algorithm, we managed to reduce the number of codes requiring expert review by 72 percent. while clinical quality measurement and pragmatic clinical trials are distinct activities, they both rely on code sets to identify their relevant subpopulations of patients, and there is clear overlap in the function between the phenotype definition code sets and quality measurement value sets. for example, there is a phenotype code set for colon cancer in the nih collaboratory, and a quality measure value set for malignant neoplasm of colon, and both have exactly the same icd-9-cm codes. because of this, we have included the quality measure value sets as an additional evaluation of the mapping methods. for the quality measure value sets, the performance of the four mapping methods followed essentially the same trend as in phenotype code sets. based on the overall f - scores, fbm performed best, followed by sm, tm, and sfm. the more aggressive methods (sm and tm) resulted in only marginal increase in recall with considerable drop in precision. therefore, if there is a need to use the gems to translate icd-9-cm code sets for clinical quality measurement, it would seem appropriate to use the fbm mapping method. note that mapping performance is generally better with smaller value sets. one possible explanation is that smaller value sets may involve more distinct and homogenous conditions (e.g., malignant neoplasm of colon, 13 codes), and that larger value sets tend to be more heterogeneous (e.g., immunocompromised conditions, 149 codes). as we found in cohort definitions, generally, for small value set (fewer than 20 codes) involving homogeneous conditions that are not in the chapters known to have undergone major reorganization in icd-10-cm (e.g., mental disorders), the fbm mappings are expected to perform very well, and only minimal manual review will be required. the existence of code sets used for phenotype definition and quality measurement raises the interesting possibility of cross - fertilization. it is conceivable that, in some cases, the same set of codes can serve both functions, as in the colon cancer example above. indeed, the icd-10-cm codes in the colon cancer value set are all considered appropriate for phenotype definition by the reviewers. so instead of defining new icd-10-cm code sets from scratch, the researchers may be able to find quality value sets defined with icd-10-cm codes that they can reuse. however, to do that one has to search through the thousands of value sets in vsac. to narrow the search, one can use some similarity measure (e.g., jaccard coefficient) between the icd-9-cm phenotype code sets and icd-9-cm value sets in vsac.30 in the future, this kind of cross - fertilization between various secondary uses of clinical codes will become more important and perhaps encourage health care organizations to participate in pragmatic trials and nationally coordinated biomedical and health services research, such as hmorn and the patient centered outcomes research network (pcornet). the phenotype knowledge base (phekb)31 and other repositories of phenotypes should consider partnerships with vsac and investigate formal linkages between research phenotypes and quality measurement value sets. the use of common value sets for clinical research and quality measurement can enable the generation of evidence from health care organizations and facilitate the vision of learning health care.32,33 for future work, we can explore ways to improve the performance of the mapping methods. there is additional information in the gems, such as flags for approximate or exact maps, and indicators of combination codes, which can be exploited to refine the mapping algorithms. boyd. showed that the mapping relationships for codes from different icd-9-cm chapters varied considerably.28 this is because the difference between icd-9-cm and icd-10-cm is not uniform across all medical specialties. outside the use of the gems, two additional mapping resources may be worthy of consideration. first, the international health terminology standards development organisation (ihtsdo) publishes a map from systematized nomenclature of medicine clinical terms (snomed ct) to icd-9-cm, and the nlm publishes a map from snomed ct to icd-10-cm. therefore, it is possible to map from icd-9-cm to icd-10-cm using snomed ct as an intermediary. second, the unified medical language system (umls) has been found to be useful in interterminology mapping.34,35 mapping relations between icd-9-cm and icd-10-cm can be discovered by exploring the synonymy and other relationships within the umls. these relationships can then be used to corroborate or supplement the maps derived from the gems. in the future snomed ct is a better clinical terminology than icd because of its coverage, granularity, clinical orientation, and logical underpinning.36 many quality value sets are already defined in snomed ct codes. although it is true that icd codes are more commonly found in ehrs at present, snomed ct codes will become more ubiquitous with the meaningful use initiative. the collaboratory demonstration projects we used were a convenience sample and are not representative of all pragmatic trials. the phenotype definitions in this study were developed to support a number of purposes for very specific research studies and might not be generalizable or appropriate for other research or quality measurement use cases related to those conditions. although we did use two reviewers for each mapping relationship, the reviews by clinical experts have not been independently corroborated. in the future for example, medication or laboratory values can be used to identify more records for potentially complete recall sets. a future related activity would be to examine a known subset of patients with chronic diseases before and after the icd-10-cm transition, and to contrast the assigned icd-10-cm codes with historical icd-9-cm codes. obviously, any practical migration of phenotyping algorithms from icd-9-cm to icd-10-cm will ultimately require the use and synthesis of other data types for validation and human review, including a more rigorously defined characterization of a gold standard diagnosis. even with rigorous gems mapping approaches, real - world application would require some level of human review to consider phenotype definitions fully validated. it is important to mention that although we focused our investigation on the translation of icd - based phenotype definitions, most phenotyping methods include other data, such as laboratory test results, medications, demographics, and natural language processing, in addition to diagnostic code value sets. while most researchers recognize that icd code sets by themselves are not sufficient for research phenotyping, these codes are widely used and remain an important component of virtually all of the phenotype definitions posted on phekb. given the national impact of implementation to icd-10-cm in october 2015, specific scrutiny of public gems tools is warranted to clarify the research implications of the icd-9-cm to icd-10-cm transition. the transition from icd-9-cm to icd-10-cm creates a heavy burden of code translation for clinical researchers using icd codes in identifying patient cohorts based on clinical criteria. although national reference mappings and tools exist to support icd-9-cm to icd-10-cm conversion, their use is not straightforward. different approaches yield different sets of icd-10-cm codes, and users should be aware of the pros and cons of each approach. in most cases, automatic code translation is not accurate enough on its own, and should be used as an auxiliary tool to assist human reviewers. variation in the migration of phenotype definitions can have an impact on the consistency of definition of cohorts and data collection over time, and can potentially have an impact on study findings if not addressed. | background : the national mandate for health systems to transition from icd-9-cm to icd-10-cm in october 2015 has an impact on research activities. clinical phenotypes defined by icd-9-cm codes need to be converted to icd-10-cm, which has nearly four times more codes and a very different structure than icd-9-cm.methods:we used the centers for medicare & medicaid services (cms) general equivalent maps (gems) to translate, using four different methods, condition - specific icd-9-cm code sets used for pragmatic trials (n=32) into icd-10-cm. we calculated the recall, precision, and f score of each method. we also used the icd-9-cm and icd-10-cm value sets defined for electronic quality measure as an additional evaluation of the mapping methods.results:the forward - backward mapping (fbm) method had higher precision, recall and f - score metrics than simple forward mapping (sfm). the more aggressive secondary (sm) and tertiary mapping (tm) methods resulted in higher recall but lower precision. for clinical phenotype definition, fbm was the best (f=0.67), but was close to sm (f=0.62) and tm (f=0.60), judging on the f - scores alone. the overall difference between the four methods was statistically significant (one - way anova, f=5.749, p=0.001). however, pairwise comparisons between fbm, sm, and tm did not reach statistical significance. a similar trend was found for the quality measure value sets.discussion:the optimal method for using the gems depends on the relative importance of recall versus precision for a given use case. it appears that for clinically distinct and homogenous conditions, the recall of fbm is sufficient. the performance of all mapping methods was lower for heterogeneous conditions. since code sets used for phenotype definition and quality measurement can be very similar, there is a possibility of cross - fertilization between the two activities.conclusion:different mapping approaches yield different collections of icd-10-cm codes. all methods require some level of human validation. |
primary aldosteronism is characterized by hypertension, hypokalemia, and sodium retention resulting from chronic oversecretion of aldosterone. in most cases, primary aldosteronism is due to a unilateral adrenal adenoma (6575%), an idiopathic bilateral adrenal hyperplasia (33%), or in rare cases an adrenal carcinoma or aldosterone - producing ovarian cancer. these cases typically present with symptoms of muscle weakness, paralysis, polyuria, headache, and sensory disturbances. the clinical symptoms associated with hypokalemia include muscle weakness, paralysis, rhabdomyolysis, arrhythmia, impaired urinary concentration, and hyperglycemia. therefore, most patients with primary aldosteronism are admitted to hospitals for headaches related to hypertension or symptoms associated with hypokalemia. we report a rare case of a patient with primary aldosteronism who presented with hypokalemia and diabetes insipidus (di). interestingly, this patient was found to have a water channel aquaporin-2 (aqp2) deficiency during the immunohistochemical examination of the kidney. we also reviewed several reports of primary aldosteronism associated with either hypokalemia or acquired nephrogenic di. a 37-year - old man with a history of hypertension was admitted to the emergency department for symptoms of muscle weakness and paralysis. he had been suffering from muscle weakness and paralysis for several years. on examination, his blood pressure was 150/90 mmhg and his heart rate was 64 beats / min. his laboratory blood chemistry results revealed a low potassium level, na 147 mmol / l, k 1.91 mmol / l, cl 108 mmol / l, and total co2 27.8 mmol / l. however, calcium (8.7 mg / dl), magnesium (2.2 mg / dl), and phosphorus (3.8 mg / dl) were within normal levels. ph 7.435, pco2 46.5 mmhg, po2 87.7 mmhg, hco3 30.5 mmol / l, o2 saturation 97.1%which indicated metabolic alkalosis. other studies included bun / creatinine level of 11/0.7 mg / dl, and the urinary chemistry profile showed a high potassium level of 18.56 mmol / l, a low urine osmolality of 199 mosm / kg, and a transtubular potassium concentration gradient of 20. on the 2 day of hospitalization, the collected 24-hour urine test revealed the following values : creatinine 317 mmol / d, urea nitrogen 2,834 mg / d, sodium 322 mmol / d, potassium 88 mmol / d, calcium 379 mg / d ; moreover, proteinuria was not detected. his plasma aldosterone (pa) level was 224.7 pg / dl and the plasma renin activity (pra) was as low as 0.11 ng / ml, resulting in a high pa / pra ratio. even though a saline loading test showed an intermediate aldosterone level (7.68 ng / dl), a left adrenal mass measuring 2 cm was observed in abdominal computed tomography (ct ; fig. although the patient had not noticed any polyuria, his urinary volume was > 3 l / d during his admission. his antidiuretic hormone (adh) level rose (224.7 pg / ml). his brain magnetic resonance imaging result showed a normal pituitary gland, and he had an insignificant response to the water deprivation test after stimulation by vasopressin injection. the patient received potassium infusions, and his potassium level improved from 1.91 mmol / l to 3.86 mmol / l. finally, a laparoscopic - assisted left adrenalectomy was performed, and the corresponding biopsy revealed it to be an adenoma (fig. 2). we also needed to prove that aldosteronism, hypokalemia, and di occurred sequentially. according to marples, prolonged hypokalemia resulted in the downregulation of aqp-2 expression in a rat kidney. however, this has not been demonstrated in humans ; therefore, a renal biopsy was also performed during the laparoscopy to investigate potential renal pathology. many proximal tubules were flattened with a loss of brush borders and were detached from the tubular basement membranes. the others had markedly swollen cytoplasm, resulting in luminal narrowing, in which brush borders were well preserved. the interstitium was markedly edematous and infiltrated by lymphocytes. in terms of water channels in the kidney, the immunohistochemistry showed that aqp-2 expression was significantly decreased, but that of other aqp channels, aqp-1 or aqp-3, was not different compared to that in a healthy kidney (fig. the serum potassium level normalized to 4.5 mmol / l without replacement of potassium, the pa level decreased to 26.7 pg / dl, and pra increased to 0.37 ng / ml, resulting in a lower pa / pra ratio. urine output also decreased to 1 cm on ct scan, then laparoscopic adrenalectomy can be considered, and adrenal venous sampling can be bypassed. by contrast, acquired nephrogenic di results from a decreased expression of aqp-2, which leads to impairment of urinary concentration. (water channel proteins) are located in the principal cells of the collecting duct, and they regulate water permeability. when adh binds to type 2 vasopressin receptor on the basolateral membrane of the principal cell, aqp-2 water in the apical membrane enters through aqp-2 and exits into the basolateral membrane through aqp-3 and -4. hypokalemia seems to cause a reduction in the intracellular level of camp, which acts as the second messenger for adh. this mechanism is similar to that of lithium - induced nephrogenic di ; however, hypokalemia - induced di is believed to have a relatively modest effect compared with lithium, and complete recovery is possible when hypokalemia is corrected. several cases complicated with secondary hypertension by adrenal adenoma have been reported in korea : aortic dissection, one of severe left ventricular septal hypertrophy, basal ganglia intracranial hemorrhage, and two cases of preeclampsia,,,. moreover, hypokalemia due to hyperaldosteronism has been reported in several case studies with periodic paralysis, two cases of rhabdomyolysis, and one instance of cardiac arrest during adrenalectomy,,. a case of nephrocalcinosis and renal cyst chronic hypokalemia leading to acquired nephrogenic di was reported in a patient with renal artery stenosis in korea. furthermore, several cases of renal disease related to hypokalemic nephrogenic di, without confirmed hyperaldosteronism, have been reported in patients with multiple autoimmune syndrome and multiple myeloma,. interestingly, in our case, renal biopsy showed evidence of chronic renal tubulointerstitial injury and decreased expression of aqp-2 in the renal collecting tubules. in summary, the authors confirmed an adrenal adenoma in a patient with hypertension and hypokalemia using ct for investigation of the potential causes of primary aldosteronism. moreover, chronic renal tubulointerstitial injury and aqp-2 deficiency developed because of chronic hypokalemia associated with primary aldosteronism. | aldosterone - producing adrenal adenoma can induce various clinical manifestations as a result of chronic exposure to aldosterone. we report a rare case of a 37-year - old man who complained of general weakness and polyuria. he was diagnosed with aldosterone - producing adrenal adenoma and nephrogenic diabetes insipidus. aldosterone enhances the secretion of potassium in the collecting duct, which can lead to hypokalemia. by contrast, nephrogenic diabetes insipidus, which manifests as polyuria and polydipsia, can occur in several clinical conditions such as acquired tubular disease and those attributed to toxins and congenital causes. among them, hypokalemia can also damage tubular structures in response to vasopressin. the patient s urine output was > 3 l / d and was diluted. owing to the ineffectiveness of vasopressin, we eventually made a diagnosis of nephrogenic diabetes insipidus. laparoscopic adrenalectomy and intraoperative kidney biopsy were subsequently performed. the pathologic finding of kidney biopsy revealed a decrease in aquaporin-2 on immunohistochemical stain. |
dietary supplement intake, in addition to training, appropriate nutrition, and advantageous gene polymorphisms, is recognized to confer exercise performance benefits [14 ]. such supplements (e.g., caffeine, bicarbonate, creatine, and beta alanine) are widely consumed by athletes in many sports. the role of caffeine as an ergogenic aid the study by costill. was the first to demonstrate improved time - to - exhaustion (tte) exercise with acute preexercise ingestion of caffeine. subsequent investigations reported that caffeine intake can improve cycling [6, 7 ], high intensity running [8, 9 ], and repeated sprint running performance. a recent review on the effects of caffeine on exercise performance suggested that, at least in endurance sports, even small to moderate caffeine doses (2 - 3 mg / kg of body mass) are likely to confer performance benefits. caffeine induces numerous physiological effects. although its stimulating effects on exercise performance in humans were initially thought to occur via increased fatty acid oxidation rates and muscle glycogen - sparing effect [5, 7 ], several studies reported conflicting findings [12, 13 ]. in addition to peripheral actions, caffeine was shown to reduce perception of effort during exercise, suggesting that some ergogenic effects may be partially accounted for by caffeine - mediated maintenance of central nervous system (cns) drive. specifically, meeusen. implied that the performance - enhancing effects of caffeine are likely to be linked with the antagonism of adenosine receptor signalling in the brain. beetroot juice consumption has recently drawn attention of many endurance athletes as evidence accumulates indicating its performance - boosting effects. several independent research groups have demonstrated in double - blind, placebo - controlled studies that acute as well as chronic intake of beetroot juice containing about 8 mmoles of nitrate (no3) can improve endurance and high - intensity intermittent performance [1620 ]. following absorption, nitrate reappears, via enterosalivary circulation, in the mouth, where it is degraded to nitrite (no2) by facultative anaerobic bacteria on the surface of the tongue. further nitrite conversion to nitric oxide (no) takes place in stomach. although the exact mechanisms by which increased beetroot juice - derived no2 and no bioavailability enhance exercise performance remain to be fully elucidated, improved muscle mitochondrial function and/or reduced high - energy phosphate turnover leading to a decreased oxygen cost of exercise are likely to be involved [16, 19 ]. considering that acute preexercise ingestion of caffeine and nitrate - rich beetroot juice has been found to enhance exercise performance via different mechanisms ; their combined effects on performance could potentially be additive. in other words the preexercise ingestion of beetroot juice and caffeine could improve performance more than intake of either beetroot juice or caffeine alone. there are relatively few studies on the effects of combining different dietary ergogenic aids, and to our knowledge, no study has investigated the potential combined ergogenic effects of nitrate and caffeine. therefore, the primary aim of this randomised, double - blind, placebo - controlled study was to evaluate potential additive effects of acute preexercise beetroot juice and caffeine supplementation on time to exhaustion during cycling at 80% vo2max in healthy, well - trained male participants. a secondary aim was determine if nitrate 's effects on the oxygen cost of exercise are influenced by caffeine co - ingestion. we hypothesised that the combination of ingesting both beetroot juice and caffeine before exercise would have greater performance enhancing effects than beetroot juice or caffeine alone. fourteen well - trained male participants volunteered to participate in this randomized, double - blind, placebo - controlled design study. their age, height, body mass, and vo2max were 22 3 years, 177 1 cm, 76 7 kg, and 63 10 ml / kg / min, respectively (values are expressed as mean sd). all participants engaged in endurance sports at least 3 times per week and all were familiar with cycling. all participants were fully informed of experimental procedures, associated risks, and a rationale of the study before they gave their written consent to participate in this study. participants were included if they were nonsmokers, currently healthy, had been training for at least 2 h per week of moderate to vigorous physical activity, and had no known history of cardiac, hepatic, renal, pulmonary neurological, gastrointestinal, haematological, or psychiatric illness. all exercise tests were performed under controlled environmental conditions (22 1c, 5060% relative humidity). approximately one week before supplementation trials participants visited the laboratory to undertake screening, vo2max determination, and familiarization tests. upon arrival, participants ' weight was measured to the nearest 0.1 kg using a digital scale (seca 770, seca ltd, germany). each subject performed a continuous incremental exercise test on one of the two electromagnetically braked cycle ergometers (lode excalibur sport, groningen, the netherlands and lode corvial, groningen, the netherlands) to volitional exhaustion to determine vo2max. subjects were randomly assigned to either one of the two cycle ergometers and the allocation was maintained during the supplementation trials. a fan was set approximately 2 m away from participants to provide cooling during exercise and supplementation trials. self - selected saddle and handlebar settings used were replicated for all subsequent supplementation trials. participants began cycling at 95 w, with increments of 35 w every 3 min until volitional fatigue despite strong verbal encouragement. samples of expired gas were collected in douglas bags during the third minute of each work rate increment, and heart rates were measured continuously using short - range telemetry (polar, kempele, finland). a paramagnetic oxygen analyser (servomex 1420b, crowborough, uk) and an infrared carbon dioxide analyser (servomex 1415b) were used to determine percentages of expired oxygen and carbon dioxide. these were used together with a dry gas meter (harvard apparatus, edenbrige, uk) for a determination of rates of oxygen consumption (vo2) and carbon dioxide production (vco2). maximal oxygen consumption was determined from the expired gas sample collected during the final minute of the exercise test when subjects indicated that they could only continue exercise for an additional minute. work rates corresponding to 60% and 80% vo2max were interpolated from the vo2-work rate relationship. after 30 min recovery participants completed a 30 min familiarization trial at 60% vo2max, which followed the same format as the experimental trials. this was undertaken to ensure that the subjects were accustomed to the procedures used during the investigation and to minimise any potential learning or anxiety effects. following completion of the preliminary tests each participant visited the laboratory on four occasions interspersed by at least four days to complete 30 min exercise at 60% vo2max immediately followed by a time - to - exhaustion trial at 80% vo2max. the treatments on these trials were as follows : (1) placebo (pla), (2) placebo + caffeine (pla+c), (3) beetroot juice (br), and (4) beetroot juice + caffeine (br+c). the treatments were administered using a randomized double - blind, placebo - controlled study design. a br+pla trial was not included because that would have supplied only half of the required amount (8 mmoles) of nitrate. on test days, participants arrived at the laboratory after an overnight fast in a rested state avoiding strenuous physical activity the day before at either 09:00 or 11:30. after ~10 min rest they consumed a standardized breakfast containing 127 kcal (24 g of carbohydrate, 2.8 g of fat, and 1.6 g of protein) and a 70 ml concentrated beetroot juice (beet - it, james white drinks ltd, ashbocking, uk) containing 4 mmol no3 or placebo (nitrate - depleted beet - it containing 5 cups of tea, coffee, and coke per day) and nitrate - rich foods (e.g. spinach, rocket, lettuce, and radish) were not permitted over the course of the study. saliva samples, for salivary cortisol, nitrite, and nitrate determination, were collected before supplement ingestion, prior to exercise, during the last 5 min of the ride at 60% vo2max, and 10 min after completion of the time - to - exhaustion test. water intake was not permitted 5 min prior to saliva collection at any time point to avoid salivary analyte dilution. briefly, participants were seated and provided an unstimulated saliva sample by passive dribble into a 7 ml polypropylene collection tube (sterilin, caerphily, uk) over a course of 24 min with opened eyes, slightly bent forward with minimal orofacial movement. all samples were immediately centrifuged at 12 000 rpm for 2 min and the supernatant was collected and stored at 20c for further analysis. the salivary cortisol concentrations were determined in duplicate using an elisa kit (salimetrics, state college, pa, usa). duplicate salivary nitrate and nitrite were analysed using a colorimetric assay kit (cayman chemicals, usa). the intra - assay coefficient of variation for cortisol, nitrate, and nitrite was 0.05). no significant differences (p 0.104) between treatments were found in any other measured variable as shown in tables 2 and 3. resting preexercise salivary nitrite and nitrate concentrations were higher as shown by the paired t - test and wilcoxon signed rank test after beetroot juice ingestion (p 0.002) than before (figures 1(a) and 1(b)). on the contrary, no differences in salivary nitrate and nitrite were found between preingestion and preexercise samples in pla and pla+c (p > 0.05). no significant differences in time - to - exhaustion protocol (f(3,39) = 2.269, p 0.096) between trials were found (figure 2) ; however, exercise time in br + c tended (p = 0.096) to be higher than in pla (1463 774 versus 1003 480 s), corresponding to a 46% improvement. similarly, 18% and 27% nonsignificant time - to - exhaustion improvements were observed on br+c trial compared with br and pla+c alone, respectively. the individual performance data demonstrate that eleven, nine, and eight subjects of fourteen cycled longer on br+c compared with pla, br, and pla+c, respectively (figure 3). there was a main effect of time with salivary cortisol being higher during exercise compared to preexercise (p 0.05). the main purpose of the present study was to evaluate potential additive effects of beetroot juice and caffeine co - ingestion on tte exercise performance. the other main goal of the experiment was to test the hypothesis if dietary nitrate reducing effects on oxygen uptake are influenced by caffeine ingestion. the major finding of this study was an 18% and 27% nonsignificant improvement in tte at 80% vo2max on br+c compared to br and pla+c, respectively. this finding supports our hypothesis implying potential positive and additive effects of br+c on exercise capacity in well - trained male athletes. to our knowledge, this is the first study demonstrating potential beneficial effects of combined acute preexercise intake of beetroot juice and caffeine on tte performance. our findings show that acute preexercise co - ingestion of beetroot juice and caffeine results in lower rpe, providing a potential mechanism, by which br+c may improve exercise performance. reduced rpe in br+c compared to pla and pla+c suggests that beetroot and caffeine co - ingestion postponed the development of exercise intolerance and/or reduced perception of effort during exercise. as this finding was not observed for the pla+c treatment, it is plausible that the reduction in rpe represents a br+c - specific effect. the physiological importance of this finding is supported by the doherty and smith meta - analysis indicating that caffeine - mediated improved exercise capacity can be largely explained by reduced rpe. preexercise caffeine intake alone has been suggested to activate neuroendocrine responses (e.g., noradrenaline secretion) to promote lipolysis and a glycogen - sparing effect [7, 10 ]. results from studies using femoral arterial - venous catheters and muscle biopsy samples before and after exercise have called this caffeine - mediated glycogen - sparing effect into question [26, 27 ]. the findings of our study support this view. we did not observe any difference in salivary cortisol between trials, nor did we find any difference in whole - body carbohydrate or fat oxidation rates. these findings imply that acute preexercise caffeine intake alone or in combination with beetroot juice does not affect neuroendocrine responses and whole - body metabolic substrate utilisation during exercise in humans. alternatively, a recent study indicated that caffeine may improve interstitial potassium handling during exercise, which is suggested to play a crucial role in fatigue development [10, 28 ]. therefore, it can be argued that caffeine may have also acted via another peripheral mechanism that we did not measure. the findings of the present study further support the ergogenic effects of beetroot juice intake on exercise performance in humans. for instance, in a randomized, cross - over design study, acute consumption of beetroot juice resulted in improved 4 km and 16 km time trial performance in club - level competitive cyclists. others have found that acute and chronic beetroot juice intake improves high - intensity tte performance, while reducing oxygen uptake [22, 23 ]. collectively, these findings suggest that both acute and chronic beetroot juice intake improve capacity to perform exercise in humans. beetroot juice - derived nitrate, nitrite, and no are thought to act via multiple mechanisms. dietary nitrate supplementation was shown to reduce the oxygen cost of exercise during both low- and moderate - intensity exercise in humans. using p magnetic resonance spectroscopy (mrs) chronic dietary nitrate supplementation attenuated the reduction in high - energy phosphate concentration (phosphocreatine) and reduced atp turnover in humans during low- and high - intensity knee - extensor exercise, suggesting that the lower oxygen cost of exercise may be, at least partially, explained by a reduced atp cost of muscle contraction. in another study, larsen. three days of sodium nitrate intake resulted in increased maximal atp production by isolated mitochondria and reduced mitochondrial oxygen affinity. interestingly, during submaximal exercise, the nitrate supplementation treatments displayed lower oxygen consumption and higher rer suggesting more efficient energy utilisation. in mice, 7 days of sodium nitrate supplementation was shown to increase intact muscle fibre force production via greater intramuscular calcium concentration, suggesting another potential mechanism by which muscle performance may be enhanced. taken together, these findings suggest that increased nitrate bioavailability is involved in the regulation of intramuscular calcium handling, high - energy phosphate utilization, and oxidative metabolism. more recently, peacock. reported that acute preexercise potassium nitrate consumption resulted in no improvement in 5 km time - trial performance in elite cross - country skiers. further, cermak. found that consumption of a single dose of concentrated beetroot juice did not affect 1 h time - trial cycling exercise performance. in a similar fashion, a 3-day period of sodium nitrate supplementation was found to cause no improvement in distance covered or mean power output over a 40 min time - trial. although these studies imply that acute and/or chronic beetroot juice intake may not confer an exercise performance benefit, a recent review by hoon. indicates that, even if not reaching statistical significance, a small positive effect on time trial and graded exercise performance may be meaningful in an elite sport context. the other main finding of the present study was that the oxygen cost of exercise at 60% and 80% vo2max on br+c was unaffected by caffeine compared with beetroot juice alone, as might have been assumed if more free fatty acids were being oxidized following caffeine consumption (i.e., oxidation of free fatty acids requires ~10% more oxygen than carbohydrate to produce the same amount of energy). this finding demonstrates that caffeine alone or in combination with beetroot juice does not affect muscle substrate utilisation and oxygen consumption during exercise. several potential factors may partially explain these discrepancies, including the exercise protocols used, participants ' fitness levels, and the form of nitrate provided. although the overall magnitude of exercise improvement on br+c may suggest physiological significance, no statistical difference between treatments was detected. this discrepancy may be, in part, explained by the study design, and thus statistical methods used to test for differences, and high interindividual variability in the tte protocol. the individual performance data demonstrated that eleven out of fourteen and nine out of fourteen participants improved their exercise capacities on br+c versus pla and br, respectively, suggesting a consistent general trend for performance enhancement on br+c, yet a range of 2613 sec was observed between the longest and the shortest br+c trial. this demonstrates that despite a general tendency for performance improvement, highly variable responsiveness exists between individual participants. this high interindividual variability may be, at least, partially explained by tte protocol, in which participants were shown to display high test - retest variability. for instance, up to 27% versus < 4% day - to - day variability has been reported for tte and time - trial protocols, respectively, suggesting that further research is warranted to fully clarify effects of combined beetroot and caffeine intake on human exercise performance. in summary, acute preexercise beetroot juice co - ingestion with caffeine augmented time - to - exhaustion at 80% vo2max performance by 18% and 27% over beetroot and caffeine alone, respectively. moreover, 46% enhancement in exercise capacity was observed in br+c compared with placebo, likely attributable to lower rpe in br+c trial. there appears to be a strong likelihood that the ergogenic effects of dietary nitrate and caffeine are additive. therefore, endurance athletes may benefit from this preexercise acute nutritional strategy prior to participation in competitive endurance events. | aims. to evaluate the possible additive effects of beetroot juice plus caffeine on exercise performance. methods. in a randomized, double - blinded study design, fourteen healthy well - trained men aged 22 3 years performed four trials on different occasions following preexercise ingestion of placebo (pla), pla plus 5 mg / kg caffeine (pla+c), beetroot juice providing 8 mmol of nitrate (br), and beetroot juice plus caffeine (br+c). participants cycled at 60% maximal oxygen uptake (vo2max) for 30 min followed by a time to exhaustion (tte) trial at 80% vo2max. saliva was collected before supplement ingestion, before exercise, and after the tte trial for salivary nitrate, nitrite, and cortisol analysis. results. in beetroot trials, saliva nitrate and nitrite increased > 10-fold before exercise compared with preingestion (p 0.002). tte in br+c was 46% higher than in pla (p = 0.096) and 18% and 27% nonsignificant tte improvements were observed on br+c compared with br and pla+c alone, respectively. lower ratings of perceived exertion during tte were found during 80% vo2max on br+c compared with pla and pla+c (p < 0.05 for both). conclusions. acute preexercise beetroot juice coingestion with caffeine likely has additive effects on exercise performance compared with either beetroot or caffeine alone. |
in the neonatal intensive care unit (nicu) population, hyponatremia is the most frequent encountered water and salt abnormality. with its broad differential diagnosis, the most frequent causes are renal salt loosing through an immature kidney and the use of drugs such as diuretics. hypertension, however, is a relatively rare feature in children, especially in neonates, with an incidence in nicu 's ranging from 0.7% to 3.2%, and renal arterial thrombosis following umbilical arterial catheterization as the leading cause [2, 3 ]. a rare clinical presentation of unilateral renal arterial stenosis is the hyponatremic hypertensive syndrome (hhs), characterized by activation of the renin angiotensin aldosterone (raas) system in the ischemic kidney, causing hypertension, and a counteracting effect on the other kidney, by different mechanisms leading to volume depletion and loss of electrolytes. this syndrome is caused by unilateral renal ischemia, due to stenosis or occlusion of a (branch of a) renal artery, and also occurs in a variety of other underlying disorders. so far, only a few reports of hhs in children are available, with polydipsia, polyuria, enuresis, weight loss, volume depletion, and various neurological and behavioural symptoms as presenting symptoms. we present a case of hhs in a preterm infant, with an extremely low sodium concentration, and discuss the difficulties encountered in treatment and the irreversible neurological sequels due to this potentially life - threatening metabolic disturbance. a preterm boy presented with extreme hyponatremia (plasma sodium of 101 mmol / l) at the 20th day after birth. he was born from a nulliparous woman at a gestational age of 31 weeks and 4 days after an uncomplicated pregnancy, followed by spontaneous rupture of membranes and antenatal corticosteroid administration. apgar scores were 9 and 10 at 1 and 5 minutes, and the birth weight was 2080 grams. an umbilical arterial catheter was inserted directly after birth, for the purpose of blood pressure monitoring, and removed after 3 days. furthermore an umbilical venous catheter and subsequently a peripheral central venous catheter were inserted for the purpose of parenteral feeding. routine cerebral ultrasonography showed an image consistent with the gestational age and mild periventricular flaring. he gained weight (from 1900 grams at the 3rd day to 2100 grams at two weeks after birth). at the age of 3 weeks rejection to feeding (until this moment consisting of 150 ml / kg / day breast milk with breast milk fortifier), weight loss (to a minimum of 1960 grams), irritability, hyperthermia, and cerebrospinal fluid analysis showed 219 leukocytes / mm with 12000 erythrocytes / mm, after a traumatic lumbar puncture, thus a meningitis could no be excluded and intravenous antibiotics were started. intravenous fluids, with a total volume of 150 ml / kg / day, containing 8 mg / kg / min glucose and 5 mmol / kg / day sodium, were administered in the regional hospital for 2 days (before return to the nicu). the plasma sodium level had declined, from 140 mmol / l nine days before, to 101 mmol / l. there were no sodium levels examined in the interval between, but the level at the onset of symptoms was established at 112 the boy returned to the nicu under suspicion of a syndrome of inappropriate antidiuretic hormone secretion (siadh) associated with the assumed meningitis, with initiated fluid restriction and sodium supplementation considered to be the appropriate therapy. we saw a pale, irritated neonate with tachypnea, arterial hypertension (104/60 mmhg, mean 78 mmhg), opisthotonus, and abnormal synchronized extensions of arms and legs. mmol / l, laboratory analysis revealed a mild hypokalemia, hypochloremia, and hypomagnesemia, with normal calcium and phosphate levels (for detailed information on all important laboratory results, see table 1). plasma osmolarity was 219 mosmol / kg, and urea and creatinine levels were normal. infection parameters were low, but liver enzymes and lactate were elevated (in the blood drawn several shortly after the seizure). furthermore an elevated plasma b - type natriuretic peptide (bnp) of 1228 pmol / l was found (in children, there are no validated data on normal values available). urinalysis showed no leucocytes, mild hematuria, low sodium and potassium, with proteinuria, glucosuria, and a urine osmolarity of 129 mosmol / kg. the combination of hyponatremia and hypertension (defined as a mean blood pressure of > 2 standard deviations for age and weight, in this case > 75 mmhg) was suggestive of renal pathology. abdominal doppler ultrasound showed a right renal arterial thrombosis, partially calcified, and an oedematous appearance of the left kidney. it was suggested that the symptoms of this neonate resulted from an hhs secondary to a renal arterial thrombosis. blood pressure levels further increased in the first hours to a maximum of 108/62 (mean 96) mmhg. we chose to carefully normalise the blood pressure with intravenous dihydralazine, causing the right kidney to become completely afunctional (as demonstrated with 99 m technetium mag3 renography in combination with the findings on doppler ultrasound as mentioned before). plasma sodium levels rose to, relatively fast within the first hours, above 120 mmol / l, and more slowly within the next 24 hours to normal. the dihydralazine and sodium supplementation could be gradually stopped after a few days, after which a normal blood pressure and electrolyte levels were maintained without any additional therapy, also urinalysis returned to normal. within a few minutes after return to the nicu the boy developed convulsions, cerebral ultrasound and magnetic resonance imaging (mri) showed extensive white matter abnormalities and the presence of a sinus thrombosis of the superior sagittal, straight, and transverse sinus. additional genetic screening revealed a mutation in the methylenetetra - hydrofolate reductase (mthfr) gene. follow - up mri at one and two months of age showed extension of the white matter abnormalities, with secondary haemorrhage, vacuolisation, and cyst formation. the gyration and myelinisation had increased, there were no signs of new ischemia, and all sinuses were recanalized. the child showed abnormal neurological behaviour (agitation, uncontrolled movements, and delayed motor development) at three months of followup. the parents of this child gave their informed consent to publication of this case report. in this case report, we describe a 3-week - old preterm boy, with extreme hyponatremia, hypertension and a dramatic neurological outcome, as a result of hhs following umbilical arterial catheterization. the typical combination of symptoms in hhs was first described in 1952 in adults, and the term hhs was established by brown. in 1965. the syndrome is not encountered frequently and in neonates it is even more rare, with renal arterial stenosis following umbilical arterial catheterization being one of the described causes [810 ], accompanied by renal microthrombi in sepsis and an association with dexamethasone use. the syndrome has been described more often in preterm than in term infants [811 ] and sometimes showed a lethal course [12, 13 ]. the high incidence of hyponatremia (30%) reported in hypertensive neonates, suggests that hhs is probably more common than we think. hhs is thought to be due to a complex interplay of different mechanisms, with unilateral renal hypoperfusion and a counteracting effect of the contralateral normal kidney as major hallmarks (two - kidney - one - clip hypertension) (figure 1). the renal arterial thrombosis causes hypoperfusion of one kidney, which activates the raas system to cause hypertension. the contralateral nonstenotic kidney reacts to this hypertension by excreting water and sodium (pressure diuresis and natriuresis) [12, 15 ]. the hypertension additionally stimulates the cardiac atrial natriuretic peptide (anp) and bnp to excrete more sodium and protein. the resulting hypovolemia, probably together with an increased production of angiotensin ii, stimulates antidiuretic hormone (adh), further aggravating the hyponatremia. furthermore, aldosterone causes renal potassium loss, which in turn stimulates renin secretion, causing a vicious circle. proteinuria, glucosuria, and hypercalciuria can also be present due to glomerular hyperfiltration in hypertension, increased renin activity, and probably even more extensive tubulointerstitial involvement. in our case, the presence of an umbilical arterial catheter, and the finding of a mutation in the mthfr gene (suggested to play role in homocysteine metabolism and enhancing atherosclerosis) were thought to be contributing factors in the development of the renal arterial thrombosis. at the moment of presentation at the nicu, our patient was thought to be already beyond the natriuretic phase of hhs (probably the severe hyponatremia finally resulted in sodium retention, explaining the low urine sodium) and most likely adh had been turned on in reaction to the volume depletion. the initial sepsis - like presentation was retrospectively interpreted as the result of a combination of hypovolemia and central nervous system disturbances. the prematurity was probably a major contributing factor leading to the severe outcome in this patient. this could have led to more extreme hyponatremia because preterms have a relatively low sodium intake, reduced tubular sodium reabsorption, and decreased glomerular filtration rate, impairing free water excretion. furthermore, recognizing the nonspecific clinical symptoms of hhs can be very difficult in a neonate. the symptoms of hhs disappeared after normalising the blood pressure with a vasodilator agent (dihydralazine), causing the ischemic kidney to become nonvital by totally abrogating the blood flow, destroying the juxtaglomerular cells, and hence stopping renin production. there was a gradual decline in blood pressure in this patient, different from the potentially dangerous fall which can be seen with angiotensin - converting enzyme (ace) inhibitors. with severe volume depletion, cautious repletion is needed, which can probably also reduce arterial pressure by suppression of raas. correction of longstanding hyponatremia should be managed carefully, to minimise the risk of developing cerebral shrinking. final therapy of the underlying renal arterial stenosis was not necessary in this case, but can be achieved by balloon angioplasty, renal artery stenting, or uninephrectomy. the remarkable irreversible neurological features in this case are most likely to be the consecutive effect of a hypertensive and hyponatremic encephalopathy, aggravated by a diminished cerebral circulation due to hypovolemia and a sinus thrombosis. furthermore the convulsions could also have led to irreversible damage to the vulnerable preterm brain. previous case reports in older children mainly mention reversible neurological symptoms, and even reversible findings on computer tomography or mri associated with hhs [19, 2224 ] and only one infant dying from massive cerebral haemorrhage. first, there is a lack of (clinical and laboratory) information about the period before the patient represented with the severe hyponatremia. unfortunately, no detailed information on water balance or 24-hour urine volumes during the period in which the patient developed the hyponatremia was available. as it is especially cumbersome to collect such data in newborns, this is very rarely done. second, no data on plasma adh, rennin, and aldosterone are available to confirm our suggested diagnosis. this case was meant to describe the complex pathophysiology of hhs, and the possible misleading clinical features in a neonate. furthermore we want to underline the risk of severe irreversible neurological damage when there is a diagnostic delay. we think that in evaluating a neonate with severe hyponatremia, hhs should be considered, especially if following umbilical arterial catheterization. | objective. to report the irreversible severe neurological symptoms following the hyponatremic hypertensive syndrome (hhs) in an infant after umbilical arterial catheterization. design. case report with review of the literature. setting. neonatal intensive care unit at a tertiary care children 's hospital. patient. a three - week - old preterm infant. conclusions. in evaluating a neonate with hyponatremia and hypertension, hhs should be considered, especially in case of umbilical arterial catheterization. in case of diagnostic delay, there is a risk of severe irreversible neurological damage. |
ulcerative colitis has its onset preferentially in young people within the second to fourth decades of life, with a second onset peak when at 60 years of age and older. its incidence is increasing in developing countries, whereas rates seem to remain stable in developed countries.1,2 in a study carried out at the rectum and colon service of hospital das clinicas da faculdade de medicina da universidade de so paulo (hc - fmusp), an incidence rate of 22 new cases per year was observed, afflicting, with greater frequency, caucasian people with a higher social - cultural status.1 even though the death rate is relatively low, its high morbidity interferes significantly with the quality of life of patients, often causing social and workplace anxiety. most of the people affected can have their disease clinically controlled ; however, as ulcerative colitis progresses, it is estimated that 20 to 30% of the patients will eventually need surgical treatment,3 since clinical treatment is no longer effective. proctocolectomy with ileal pouch - anal anastomosis has revolutionized the surgical therapy of ulcerative colitis, making it the surgery of choice, especially for young adults, since it removes all of the disease and allows sphincter conservation.4 j - pouch reconstruction, introduced in 19875 and later by kiss,6 is the most commonly used reconstruction due to the volume capacity obtained, simplicity in execution, ease of reaching the anal canal, and elimination of the possibility of spontaneous transanal evacuation. furthermore, j - pouch reconstruction does not require catheterization like the s - pouch reconstruction does.4 the focus of interest is on studying the quality of life of patients who have undergone proctocolectomy with ileal pouch - anal anastomosis and assessing the systemic, emotional, and social parameters that may interfere with the day - to - day lives of these patients.7 international literature suggests that the establishment of functional parameters takes place a year after the surgery or the closing of the ileostomy, if one is used,812 allowing time for an adequate evaluation of the results after this period. the world health organization defines quality of life as the perception of the individual of his / her position in life in the cultural context and the value system in which he / she lives in relation to his / her objectives, expectations, standards, and concerns.13 in this definition, it is implied that the concept of quality of life is subjective, broad, and includes elements of both positive and negative impact. in order to study quality of life after a surgery, the inflammatory bowel disease questionnaire (ibdq) is used. it is a reliable measure, has good reproducibility, and reflects the important changes that occur in the health state of intestinal bowel disease patients. it can be used to thoroughly verify the impact, effectiveness, and efficiency of therapeutic measures. the ibdq has recently been translated and validated into the portuguese language.14 using this questionnaire at hcfmusp, we have studied the quality of life of patients who have undergone proctocolectomy with ileal pouch - anal anastomosis for ulcerative colitis after an average of five years post - operation. at that time it was observed that 73% of the patients claimed that their quality of life was considered great or good after the surgery.15 it has been published in the literature that long - term patients, 10 years or more post - operation, develop a large number of fistulas that connect the ileal pouch to the perineum and vaginal skin, as well as a large number of pouchitis.16 we are not aware of national studies (pubmed database) that evaluate the quality of life of patients who have undergone surgery ten years earlier. for this reason, we have decided to publish this study, employing the same methodology used in a masters degree that was presented to, and approved by, the gastroenterology department at fmusp, which was later published15 after the ibdq was validated in brazil. the goal of the present study was to assess the quality of life of ibd patients who have undergone proctocolectomy with ileal pouch - anal anastomosis over ten years ago at the rectum and colon services of the medical school of the university of so paulo by employing the ibdq. the study was composed of ulcerative colitis patients who underwent proctocolectomy with ileal pouch - anal anastomosis over ten years ago. these patients have been see at the rectum and colon surgery ambulatory services of the second surgical clinic division of hospital das clinicas da faculdade de medicine da universidade de so paulo since 1985. all patients who have undergone surgery at hospital das clinicas da faculdade de medicine da universidade de so paulo within the years from 1985 to 1995, who did not require an ileostomy because of post - operative complications or those whose temporary ileostomy had been closed were included in this study. the patients gave their written consent after being informed by the author of the goals, methods used, and risks and benefits of this research. patients who underwent surgery using videolaparoscopy, missed the ambulatorial follow - up, were children, and failed to understand the questionnaire were excluded from this study. the patients were interviewed during their regular visit for the post - operative observation or through requests made for an appointment via telephone contact. the author has interviewed the patients using the ibdq at the hospital das clinicas da faculdade de medicine da universidade de so paulo. the ibdq is comprised of 32 items that consider 4 areas (or dimensions) : intestinal symptoms (evacuation frequency, abdominal pain and/or cramps, degree of continence, and evacuation urgency), systemic symptoms (nausea, fatigue, insomnia, and weight loss), social aspects (working ability and social and leisure activities), and emotional aspects (behavior changes, irritability, anger, melancholy, and degree of satisfaction in relation to the surgery). the questionnaire was multiple choice, consisting of a 7-point likert scale, with 1 being the worst quality of life and 7 the best. the range of final scores, which results from adding the value of each answer, varied from 32 to 224, with the higher scores being an indicator of a better quality of life. the length of time needed to complete the questionnaire varied from 15 to 45 minutes. the patient, previously instructed on how to answer the questionnaire, chose from a chart with choices numbered from 1 to 7 the choice that best answered the question posed. the questions were repeated, so as to clarify the patient s understanding that the desired data were those from two weeks prior to the interview. clinical studies published in the literature that use the ibdq1720 simplified interpretation of the data in the questionnaire ; the control group typically scores above 200, clinically stable ibd patients score an average of 169, and currently symptomatic ibd patients score below 150. based on these studies, the following classifications for post - op quality of life were used in this study : the scoring of the questions in all realms was transformed in the following way : the 1 to 7 scale was transformed into a 0 to 1 scale such that the values of the variables were altered from qualitative ordinal to quantitative, so as to facilitate statistical analysis and figure disposition. the chi - square and kruskal - wallis tests were used to statistically assess the collected data. the pearson s chi - square test and monte carlo method were used to verify the existence of significant differences among the results of the ibdq for different age groups and men and women. for each area individually and for all of them combined, the kruskal - wallis test was used to verify if there were differences in the ibdq scoring among the groups in relation to the proportions. a significance level of p<0.008 due to the bonferroni correction (multiple comparisons) was established for all statistical tests. the study was evaluated and approved by the ethics committee of hospital das clinicas da faculdade de medicine da universidade de so paulo. between 1985 and 1995, 65 ulcerative colitis patients underwent proctocolectomy with ileal pouch - anal anastomosis at the hospital das clinicas da faculdade de medicina da universidade de so paulo. from this group, 12 patients still had an ileostomy for several reasons, 12 patients could not be reached because they lived in different states, and 5 patients had died for reasons other than the surgery. a total of 36 patients were studied, 14 (38.9%) males and 22 (61.1%) females. the average and median ages were 45 and 44 years, respectively, while ages ranged from 28 to 64 years old using the classification defined in the methodology section above for the ibdq, the quality of life was considered excellent for 9, good for 11, regular for 13, and bad for 3 patients (table 1). when comparing the ibdq classification to gender, it was observed that the good results were observed predominantly for men ; however, do not shows significant difference (table 2). in the same way, when comparing the classification to age, it was observed that the bad results were most often associated with the older patients, but these differences were also not meaningful. age was divided into two categories : older than 55 (25% of the patients) or younger than 55 (75% of the patients), where in the results are shown in table 3. by applying the kruskal - wallis test using the bonferroni correction in the comparison among the aforementioned areas, a significant difference (p - value<0.008) in the quality of life was detected when intestinal symptoms were compared to systemic symptoms (p - value = 0.006) and when systemic symptomso were compared to the social aspects (p - value = 0.007), as shown in table 1. when the questions were analyzed within their own areas so as to identify the differences between the questions and separate those with the lowest scores from those with the highest, it was found that some of them were predominant and have a profound weight in the final scoring of the ibdq. on the other hand, due to the small number of samples and great number of questions, it was not possible to make comparison inferences ; only the descriptive analysis for the study of the questions within their own areas was possible. in order to better understand the results and simplify the analysis, we named the questions by using keywords included in the questions themselves, as shown in figures 2 through 5. in the intestinal symptoms area, the lowest score was given for diarrhea (average = 0.58, median = 0.57, and standard deviation = 0.32), while the highest was given for rectal bleeding (average = 0.91, median = 1.0, and standard deviation = 0.17), as presented in figure 2. by separately analyzing the questions in the systemic symptoms area to identify questions that had the greatest weight in their answers, it was noted that an adequate night of sleep, without having to wake up due to an intestinal problem, was the question that received the lowest score (average = 0.55, median = 0.57, and standard deviation = 0.30). in contrast, physical disposition received the highest score (average = 0.68, median = 0.78, and standard deviation = 0.25), as depicted in figure 3. in the social aspects area, the questions regarding places without restrooms (average = 0.63, median = 0.71, and standard deviation = 0.35) and social appointments (average = 0.83, median = 1.0, and standard deviation = 0.23) received the lowest and highest scores, respectively (figure 4). finally, in the emotional aspects area, the questions regarding irritated (average = 0.57, median = 0.57, and standard deviation = 0.28) and pleased and thankful for his / her personal life (average = 0.85, median = 0.85, and standard deviation = 0.20) received the lowest and highest scores, respectively, as shown in figure 5. in addition, the question pleased and thankful for his / her personal life received the highest score in the questionnaire. the selection of the instrument should be based on the clarity of its components, definition of the target audience, and disease for which it was developed. in addition, this instrument should be easy to apply and easy to understand and have an appropriate time of use.21 the ibdq was used in this study since it is a specific instrument with simple language, it has a viable time of use, and it is increasingly being accepted in literature. in addition, it has been translated and validated in the portuguese language.14 the quality of life for ibd patients after the creation of an ileal pouch was similar to that of the general population, which, according to lichtenstein.,22 represents the re - establishment of a good quality of life in the postoperative stage for patients of all ages. fazio.12 observed the quality of life to be excellent or good for 93% of the 645 patients studied after 40 months post - operation. in our study, a similar result was obtained ; however, a less positive result was obtained for the long post - operative period since the patients could have presented pouchitis and other complications. for some patients, surgery could not restore adequate intestinal function and in some cases caused sexual or fertility problems. furthermore, the question about sexual activity asked in the systemic symptom section did not present a low score. the influence of age on the functionality of the ileal pouch was of constant concern in several studies comparing patients of different ages at the moment of surgery. jorge.23 studied the recovery of anal sphincter functionality in 22 patients aged 50 years and older and could not identify any differences compared to 50 young patients. nevertheless, the transitory damage of the inner anal sphincter was more severe in patients aged 50 years and older, even though there was total recovery after the closing of the ileostomy. delaney.,24 in a study composed of 1895 patients after a four - and - a - half - year follow - up, observed general satisfactory quality of life and functional results even in patients aged 65 years and older. this study, despite the seemingly bad results associated with patients aged 55 years and older, did not provide statistically significant results, showing that the long - term damage in quality of life is small. therefore, 95% to 98% of the patients in this study would have undergone the surgery again, and the same percentage would recommend it to other patients. by analyzing the questionnaire answers, 30 to 40% of the variations occasionally observed in the different sections can be explained by four independent variables : pouchitis, ileal pouch post - operative complications, extraintestinal manifestations, and possible psychiatric disorders.25 regardless of the fact that complication risks are often associated with the ileal pouch, the most frequent being pouchitis, which can persist for long periods, the chance of removing the ileal pouch seems despicable after 10 years post - operation.26 almeida.15 studied 30 patients who have undergone surgery at the rectum and colon service at hcfmusp after an average of five years post - operation, and they observed that 87% of these patients were satisfied with the surgery and that 73% reported an excellent or good quality of life. we applied the ibdq to the 30 patients previously interviewed by almeida.15 and to 6 other patients who had not been previously interviewed. in our study, we were able to determine an adequate and satisfactory quality of life, similar to that found in the literature.27,28 more specifically, 85% of the interviewed patients were satisfied and thankful for having undergone the surgery. mcleod and baxter29 stated that the satisfaction achieved from having the surgery can be determined by the understanding that the treatment carried out was far better than any other alternative. the quality of life considered excellent or good for 55.6% of the patients was lower than that formerly found in the almeida.15 study, which may be due to individually desired personal expectations, inadequate information about excessively optimistic expectations, and the extent of previously used drug dependency. in this study, as well as in almeida.,15 it could be verified that the intestinal and social areas were the areas that most influenced the results of the quality of life questionnaire. in addition, the systemic section presented the least weight in the final scoring of the ibdq in both studies. biopsychosocial reasons were related to the quality of life. even though patients reported mental and physical decay in comparison to the general population, after another assessment, they demonstrated a better result than was previously obtained. medicine and psychotherapeutic methods for pain and managing anxiety can be taken into account when treating these patients so as to improve their quality of life.25 the psychological condition of the patient becomes clear when we observe that certain answers on the questionnaire influence the final scoring, for example, an adequate night of sleep and physical disposition for day - to - day activities, among others. hahnloser.30 studied the quality of life in patients who had been observed on a yearly basis. these patients have disclosed that proctocolectomy with ileal pouch - anal anastomosis presents good functional and quality of life results and remained stable even after 15 years post - operation. this same author later presented a case study of a 20-year observation period that showed adequate and satisfactory results.31 this study analyzed 1035 patients after a 10-year period and 251 patients after a 20-year period, wherein it was demonstrated that the number of continent patients, both during the day and at night, remained relatively stable. these results notwithstanding, a decrease in nocturnal continence has been noticed. in our case study, it was noted that the lowest score on the ibdq was for the question an adequate night of sleep without having to get up due to an intestinal problem. it is worth mentioning that as the years go by, the sphincter strength declines and obstetrical trauma sequels may occur ; that is, inflammatory bowel syndrome becomes more common with age. these factors should be taken into account when loss of continence is observed after long periods of time. the personal aspects involved in answering the questionnaire (the disclosure and fear) and the sub - answer trend of the patients, as well as the small number of patients presented in this case study, may influence the results obtained. nonetheless, the results do not differ greatly from those presented in the literature. there are few reports on the quality of life of ileal pouch post - operation patients in the national literature, and none of these reports cover a period of more than five years of observation. the high level of satisfaction obtained with this questionnaire shows that the ileal pouch procedure continues to be an adequate surgery with acceptable results, as demonstrated by the highest - scored question on the ibdq. the results found by surgeons, in which the patients are pleased to avoid a stoma, makes the procedure of sphincter preservation a focal point. the functional results should not be the only parameter that influences the choice of a procedure. most of the patients who have undergone proctocolectomy with sphincter preservation and ileal j pouch - anal anastomosis over ten years ago have presented a high score in the quality of life questionnaire (ibdq). therefore, it can be concluded that the possibility of sphincter preservation should always be considered, since patients remain clinically stable and have a good quality of life even after a long period post - operation. | objectiveto evaluate, by means of the inflammatory bowel disease questionnaire (ibdq), the quality of life of ulcerative colitis patients submitted to proctocolectomy with sphincter preservation using j - pouch reconstruction over ten years ago.methodsthe study consisted of 36 patients interviewed using the inflammatory bowel disease questionnaire. the score scale, resulting from the addition of each answer, ranged from 32 to 224, where the highest score indicates the best quality of life. the chi square test was used to verify the existence of meaningful differences between the results of the questionnaire and age, and gender proportion. for each section, as well as for all of them combined, the kruskal - wallis test was used to verify if there were differences in the inflammatory bowel disease questionnaire scores among the groups in relation to the proportions.resultsafter applying the inflammatory bowel disease questionnaire, it was determined that quality of life was considered excellent for 9 (25%), good for 11 (30.6%), regular for 13 (36.1%), and bad for 3 (8.3%) patients. in our study, we determined that 85% of the patients were pleased with and thankful for the surgery that they underwent.conclusionwe can conclude that the possibility of sphincter preservation should always be taken into account, since patients remain clinically stable and have a high quality of life even after long periods. |
. they may appear either as single entity or as multiple cysts associated with syndromes like gorlin goltz syndrome. later, gorlin and goltz illustrated this lesion in detail with features such as multiple nevoid basal cell carcinomas (bccs), bifid ribs, jaw cysts, and other features ; hence, this lesion is called gorlin goltz syndrome or basal cell nevus syndrome or jaw cyst bifid rib syndrome, or multiple nevoid bcc syndrome. this lesion is inherited in autosomal dominant manner characterized by total penetrance and variable expressivity. various treatment modalities have been suggested by different authors for okcs. for small lesions enucleation after surgical enucleation, the application of carnoy 's solution has been suggested to prevent recurrences. complete treatment of syndromic okcs involve a team of dental, medical, and genetics specialties. a periodic follow - up is recommended for these lesions due to their high recurrence rates. we are presenting a case of gorlin goltz syndrome and briefly talk about its pathogenesis, diagnostic criteria, and differences between syndromic and asyndromic okcs. a 20-year - old male came to the oral medicine department with a chief complaint of swelling on lower right and left back teeth region since 2 months. the swelling was initially smaller in size and gradually increased in size to attain the present dimensions. the history of present illness revealed that there was swelling in the upper front teeth region 2 years back for which he consulted a dentist and the upper front teeth were extracted. again he visited the same dentist and during the check - up the other two swellings were diagnosed on right and left back teeth region and adjacent to extracted teeth. the patient 's past medical history revealed a history of epileptic attack 2 months back. extraorally, frontal bossing, depressed nasal bridge, ocular hypertelorism, prominent supra orbital ridge, and mild mandibular prognathism were found. there were two diffuse swellings seen on right and left side of the face at the junction of body and angle of the mandible measuring about 1 cm 1 cm in size. two diffuse swellings were evident on intraoral examination in the retromolar region of the left side from 37 region to retromolar area and on right side distal to 48 region at the level of occlusion. on palpation, all the inspection findings were confirmed and the swellings were hard and nontender with diffuse borders. missing teeth were 13, 23, 18, and 28, whereas 38 and 48 were impacted. a radiographic examination was carried out that comprised of a panoramic and periapical films, computed tomography (ct) scan of brain, and chest x - ray along with ultrasound of abdomen and pelvis. on evaluation of radiographs, it was observed that there were three unilocular radiolucencies of various sizes located at the lower right (adjacent to 48), left mandible (38), and upper left maxilla (2124) [figure 1 ]. chest x - ray revealed splaying of ribs 89, 910, and 1011 [figure 2 ]. ct scan of brain revealed calcification of falx cerebri and tentorium cerebri [figure 3 ]. ct scan showing bilateral falx cerebri calcification chest x - ray exhibiting splayed ribs with a provisional diagnosis of multiple dentigerous cysts, an enucleation of the cyst was performed. gross examination revealed multiple bits of soft tissue specimens and cystic lining, together measuring about 0.5 cm 2 cm, which are whitish to creamish in color and leathery in consistency [figure 4 ]. the cystic lining appeared as parakeratinized corrugated stratified squamous epithelium that was about 34 layer thick without any rete ridges. diffuse chronic inflammatory cells were also seen [figures 5 and 6 ]. based on the histopathological findings, a diagnosis of keratinizing cystic odontogenic tumor in relation to lower right and left posterior teeth region and upper right anterior teeth region was made. after correlating clinical, radiological, and histopathological features, a final diagnosis of gorlin goltz syndrome was given. we decided to carry on the treatment of the patient after the taking medical opinion. the patient was referred to a medical hospital to seek opinion but the patient did not turn up later. gross specimen showing multiple soft tissue thin cystic lining parakeratinized palisaded corrugated epithelium islands invading in connective epithelium gorlin goltz syndrome is a uncommon entity with multiple okcs as its first manifestation ; hence, dentists have an important role in its early detection thereby achieving proper management of this syndrome. studies have shown that it has an incidence of about 1 in 50,000150,000. apart from gorlin goltz syndrome, multiple okcs are seen in other syndromes such as ehlers danlos syndrome, orofacial digital syndrome, and noonan syndrome. the lesion is inherited in a dominant pattern. the review of the literature about its pathogenesis revealed mutation of the protein patched homolog (ptch) gene, which is mapped to 9q21 - 23 chromosome. these genetic studies suggest that in this syndrome there is an abnormal hedgehog signaling pathway. ptch acts as a receptor for sonic hedgehog gene, which has a primary role in embryogenesis. smoothened (smo) has a role in cell growth and differentiation and in hedgehog signaling pathway, hh binds to the other component patched (ptc) and releases smo. in this case, the mutated ptch gene results in abnormal hedgehog, which will not bind to ptc, which in turn inhibits smo thus affecting cell growth and differentiation and may result in abnormalities such as neoplasms and others. goltz syndrome shows a spectrum of clinical manifestations that can be broadly put in six categories [table 1 ]. goltz syndrome, two major or one major and two minor criteria should be present [table 2 ]. in case of our patient, the diagnosis was confirmed as he matched three major criteria (multiple okcs, splayed ribs, and calcified falx cerebri) and five minor criteria (frontal bossing, nasal bridge depressed, ocular hypertelorism, prominent supra orbital ridge, and mild mandibular prognathism). clinical manifestations of gorlin goltz syndrome major and minor diagnostic criterion of gorlin goltz syndrome literature review of clinical features showed that gorlin goltz syndrome is usually seen in younger age group ranging in between 10 years and 30 years. the commonest site of okcs for gorlin goltz syndrome is maxillary molar area ; our case presented with multiple cysts bilaterally at posterior mandible and ramus area and at maxillary incisor canine area. radiologic characteristics of okcs show unilocular, well - defined radiolucent lesions, usually associated with unerupted tooth. our case showed unilocular radiolucency in relation to an unerupted right and left third molars along with upper anterior teeth. generally, means the presence of more than one cyst at a time, whereas in this case multiple cysts means presence of more than one cyst in one 's life time. histologically, okcs show corrugated para- or orthokeratinized surface, almost equal uniform thickness of the epithelium, basal cells showing tomb stone or picket fence arrangement. few cases show the presence of daughter or satellite cells in the underlying connective tissue and these cysts show more recurrence rate. parakeratotic okcs are more common and more aggressive than orthokeratotic okcs. in case of gorlin goltz syndrome, parakeratotic okcs are seen. the major differences between okcs associated with gorlin goltz syndrome and solitary okcs are listed in table 3. our case showed three to four layered thick parakeratinized corrugated stratified squamous epithelium and presence of odontogenic island in underlying connective tissue. differences between syndromic okcs and solitary okcs the treatment of okcs is usually carried out by either enucleation or marsupialization. the studies have shown that multiple okcs are detected almost 10 years before the appearance of other symptoms of gorlin goltz syndrome. hence, a dentist has a very important role in treating this syndrome as he will be the first person to detect oral findings and predict occurrence of syndrome in future. comprehensive treatment of this syndrome requires a dental and medical opinion as well as genetic counseling. a rare case of gorlin goltz syndrome that showed its uniqueness in that it was seen in a male patient and the site of the cysts was bilaterally at posterior mandible and ramus area and in maxillary incisor canine area not at its usual location of maxillary molar area as seen in majority of patients. we suggest that patients with multiple okcs should be thoroughly evaluated as they are the major component of gorlin goltz syndrome and early findings of this syndrome. these patients should be followed for a long time with proper medical care and genetic counseling so as to prevent the development of other complications such as malignancies. | odontogenic keratocysts (okcs) may occur in two different forms, either as solitary (nonsyndromic okcs) or as multiple okcs (syndromic okcs). multiple okcs usually occur as one of the findings in gorlin goltz syndrome with other features such as skin carcinomas and rib, eye, and neurologic abnormalities. we report a rare case of gorlin goltz syndrome in a 20-year - old male patient who presented with a slow growing swelling on lower right and left back teeth region since 2 months. apart from these, other findings were frontal bossing, depressed nasal bridge, ocular hypertelorism, prominent supra orbital ridge, and mild mandibular prognathism. excision was done and microscopic study revealed okc and the follow - up could not be carried out for the complete management. we also presented a review of its pathogenesis, criterion, and differences between syndromic and nonsyndromic okcs and suggest to thoroughly examine any patient who presents with multiple okcs to rule out syndromic variety. |
psoriasis is a chronic, immune mediated skin disease characterized by erythematous plaques covered with a silvery - white scale, predominantly over the extensor surfaces and the scalp. males and females are equally affected by psoriasis vulgaris, with an earlier age of onset in females. however, there is often a genetic predisposition, and sometimes an obvious environmental trigger such as an infection. characteristic cutaneous disorders such as wilson 's disease and menke 's kinky hair disease are caused by abnormal copper metabolism ; acrodermatitis enteropathica is caused by zinc deficiency. some investigators have also reported low serum zinc levels in cutaneous disorders such as acne vulgaris, lichen planus, ichthyosis, etc., against this background, there is a possibility that abnormal metabolism of both these metals may also exist in psoriasis. this was a prospective hospital - based case control study involving 100 cases of psoriasis aged 18 years and above and 100 age and sex matched healthy controls. any patient having a disease or condition known to alter the levels of serum proteins, trace elements or alkaline phosphatase or receiving any systemic treatment for psoriasis for at least four weeks before enrolment were excluded. the cases comprised psoriasis patients attending the inpatient and outpatient departments of a dermatology department at a government - run medical college. the control group comprised of patients with other unrelated insignificant complaints, attendants of patients and staff members of the hospital. informed consent was taken from both groups, and data was recorded on a standard proforma. plasma levels of zinc and copper were estimated in both groups in addition to the routine investigations. the statistical analysis of the data was performed using graphpad inc. and instat 3 software. the measurements were expressed as mean standard deviation (sd). to evaluate the differences between means of the cases and controls, we used a nonparametric test, the mann - whitney test as our data was not following a gaussian distribution. the youngest patient in our study was a 19 years old male, and the oldest a 66 year old male. males predominated in our study (67%) as compared to females (33%). onset of psoriasis was commonest in the age group 21 - 30 years ; females had a younger age of onset as compared with males. the most common type of psoriasis among our patients was the chronic plaque type followed by the guttate type, with palmoplantar type being the least common. as far as the serum levels of zinc, copper, albumin, and globulin are concerned, there was a statistically significant difference in the serum levels of cases and controls. the serum levels of zinc and albumin were significantly low and levels of copper and globulin were significantly raised among cases as compared with controls. the serum levels of alkaline phosphatase were comparable in cases and controls [table 1 ]. psoriasis is a common chronic inflammatory skin disease characterized by a marked increase in keratinocyte proliferation and abnormal differentiation, prominent alterations in dermal capillary vasculature and the presence of dermal and epidermal mononuclear leukocytes and neutrophils. a complex interaction between the innate immunity, adaptive immunity and a skin barrier defect is likely to explain the possible pathogenesis of psoriasis, though the response of the disease to biological response modifiers points primarily towards the role of adaptive immunity in causation. though exact cause in not known, oxidative stress has been widely implicated in the pathogenesis of psoriasis. it has been suggested that generation of reactive oxygen species (ros) from neutrophils, keratinocytes, and fibroblasts can contribute to neutrophil activation, which plays an important role in the psoriatic process. superoxide dismutase is an important antioxidant enzyme in the body, which plays an essential role in limiting the harmful effects of ros, which are reported to have a role in causation of psoriasis. zinc and copper are an integral part of as many as 40 metalloenzymes, including alkaline phosphatase and superoxide dismutase, and changes in their serum levels may reflect in changes in the activity of these enzymes. researchers have noted that psoriatic lesions retain a high content of zinc compared with uninvolved skin suggesting an imbalance in zinc distribution between serum and psoriatic lesions. this has been reflected in some studies showing decreased levels of zinc in patients with severe psoriasis. however, some studies found no statistically significant difference in the serum zinc levels among psoriatics and the normal population. moreover, decreased serum albumin can also results in alteration of serum zinc levels in psoriasis patients, though some studies have shown that zinc levels are decreased irrespective of serum albumin levels. reduced albumin in psoriasis patients has been suggested to be due to lowered rates of albumin synthesis or increased rates of turnover. later, it was also suggested that the hypoalbuminemia in psoriasis patients may be the result of an increased endogenous catabolism of albumin without significant loss through urine, stools or skin. some researchers have even suggested an increased uptake of albumin by liver and splenic macrophages. copper, another important trace metal is present in the serum in at least two fractions, a transport fraction (5%) loosely bound to albumin and ceruloplasmin (95%) firmly bound to globulin. it is important to note that serum copper largely reflects serum ceruloplasmin and is not a sensitive indicator of copper nutritional status. serum ceruloplasmin levels are known to increase by 50% or more under certain conditions of physical stress, such as trauma, inflammation, or disease. because over 90% of serum copper is carried in ceruloplasmin, which is increased in many inflammatory conditions, elevated serum copper as seen in our study hinks. have demonstrated a similar increase in serum ceruloplasmin and copper in psoriasis. demonstrated statistically insignificant higher values of ceruloplasmin among psoriatics as compared to controls ; however, they demonstrated a higher copper / ceruloplasmin ratio in psoriatics as compared with controls, which was statistically significant. the results of this study suggest zinc and copper may have a role in etiology of psoriasis as they influence the levels of essential enzyme superoxide dismutase. however, whether these levels reflect the abnormalities caused by the disease process or are simply responsible for setting the pathogenesis of psoriasis into motion needs to be investigated further by large - scale studies. | background : psoriasis is a chronic, immune - mediated skin disease with unknown etiology, with an epidermal turnover time of < 10 days compared to a normal turnover time of 4 - 8 weeks. this epidermal hyperproliferation accounts for many of the metabolic abnormalities including alteration in the serum levels of proteins and some trace elements.aim:the aim was to detect any statistically significant difference in the serum levels of zinc, copper, albumin, globulin and alkaline phosphatase between psoriasis patients and healthy controls.materials and methods : hundred cases of psoriasis and 100 age and sex matched controls were enrolled in a hospital based case - control study. the serum levels of zinc, copper, albumin, globulin and alkaline phosphatase were calculated and compared among the cases and controls and evaluated statistically.results:serum zinc levels were significantly low in the psoriasis group as compared with controls (mean 80.028 g / dl vs. 109.179 g / dl, p < 0.0001). serum copper levels were significantly raised among cases as compared with controls (mean 167.317 g / dl vs. 133.884 g / dl p < 0.0001). serum albumin levels were significantly decreased (3.762 g / dl vs. 4.103 g / dl, p < 0.001), whereas serum globulin levels were raised (3.296 g / dl vs. 2.596 g / dl, p = 0.0014) among cases as compared with controls, respectively. serum alkaline phosphatase levels were comparable between the two groups.conclusion:the results of this study show significant alterations in the serum levels of copper, zinc, albumin, and globulin in psoriatic patients. this paper aims at highlighting the possible role of trace metals copper and zinc in the aetiopathogenesis of psoriasis and also provides a proposed interplay of factors involved in the pathogenesis of psoriasis. |
years after surgical procedures are performed, operative reports are often the only source of information another surgeon possesses when attempting to understand the history and internal anatomy of a patient. evidence shows that a structured format for documenting findings improves overall accuracy of reporting and, by extension, is likely to improve patient outcomes [1, 2 ]. an appropriately detailed report may greatly improve treatment strategy and general preparedness for a case, theoretically leading to better patient safety and care. while efforts have been made in the general surgical field to improve and standardize operative reports, these efforts are still lacking in gynecology surgery. pelvic anatomy is unique in that various pathologies can be missed if not intentionally sought out for identification. these anatomical characteristics could influence the detailed description of pelvic findings during surgery in general and, more specifically, during laparoscopy. while the false pelvis is the space enclosed by the pelvic girdle above and in front of the pelvic brim and considered part of the abdominal cavity, the true pelvis includes the genital tract midline between the lower end of the urinary tract anteriorly and the gastrointestinal tract posteriorly. the ligamentary attachments of the female genital organs add to the anatomical uniqueness of the pelvis. for instance, the round ligament, which extends from the cornua to the internal ring, could harbor pathology from its origin to its insertion. the uterosacral ligaments and the suspensory ligaments of the ovary are often inspected but not described. other anatomically obscure locations include the ovarian fossa, the lateral pelvic sidewall, and the area inferior to the uterosacral ligament. the objective of this study is to propose a method for systematic pelvic assessment based on anatomical landmarks and structured documentation with laparoscopic photography. to illustrate the current deficiencies, we retrospectively applied this system to a cohort of patients who underwent laparoscopy for unexplained infertility to assess the comprehensiveness of the operative reports. in our proposed system, the pelvis was topographically divided into two midline zones (zone i & ii) and two paired (right and left) lateral zones (zone iii & iv). zone i is the area between the two round ligaments from their origin at the uterine cornua to their insertion in the deep inguinal rings. zone ii is the area between the two uterosacral ligaments from their origin from the back of the uterus to their insertions in the sacrum posteriorly. zone iii is the area between the uterosacral ligament inferiorly and the entire length of the fallopian tube and the infundibulopelvic ligament superiorly. zone iv is the triangular area lateral to the fallopian tube and the infundibulopelvic ligament and medial to the external iliac vessels up to the round ligament (figure 1). this study was conducted at the university hospitals case medical center (uhcmc), case western reserve university, cleveland, oh, usa. after irb approval was obtained, operative reports of 540 patients who underwent diagnostic or operative laparoscopy for the diagnosis of unexplained infertility between january 2005 and january 2012 were collected. the operative reports for these patients were reviewed with allocation of the reported positive or negative findings to the respective zones as shown above. all reports were evaluated for the comprehensiveness of the description with respect to normal findings or pathology for six zones as follows. using this mapping of the pelvis, during the review period of the study, 8876 laparoscopies and hysteroscopies were performed within the entire uhcmc system for a variety of indications. of these, a total of 540 cases of diagnostic and/or operative laparoscopy with and without hysteroscopy for unexplained infertility were identified. these cases were selected as they are usually intended as a careful surveillance of pelvic anatomy in order to identify an etiology of infertility. as the goal of these surgical cases is the identification of anatomy, it was thought fit that these operative reports would focus on the description of anatomy. all operative reports commented on the uterus, tubes, and ovaries (100%), which reflect parts of zone i and part of zone iii. only 17% (n = 93, 95% ci : 13.820.2) commented on the dome of the bladder and the anterior cul - de - sac (the remainder of zone i). twenty - four percent (n = 130, 95% ci : 20.427.6) commented on the posterior cul - de - sac, which represents part of zone ii. interestingly, only one fourth of those who addressed zone ii (6% ; n = 34, 95% ci : 48) commented on the rectosigmoid. moreover, only 5% (29/540) commented on the pelvic sidewall peritoneum without specifying whether the ovarian fossa and the peritoneum overlying zone iv were evaluated. overall, only 6% (n = 34, 95% ci : 48) reported either positive and/or negative findings in the various pelvic zones resulting in complete documentation of the presence or absence of pelvic findings (table 2). supplemental photographic documentation of all pelvic areas was frequently missed ; it was found only in 6% (n = 34, 95% ci : 48) of patients ' charts. the paucity of detail in operative reporting represents a missed opportunity to document important anatomical findings that could prove useful in future patient care. our retrospective chart review demonstrated that description of important pelvic structures is frequently missing in operative notes from diagnostic and operative laparoscopy. the anterior cul - de - sac, deep inguinal rings, ovarian fossa, and the lateral pelvic sidewall peritoneum are the most frequently missed areas. as seen in the general surgical literature, standardizing operative reporting improves completeness of documentation. if such systems are in place, residents can be taught these methods for reporting during their training [3, 4 ]. as the era of digital photography and electronic medical records evolves, this is a very appropriate time to innovate with respect to the methods by which we document our surgical findings. implementation of a systematic approach for laparoscopic pelvic examination will indeed enhance the diagnostic accuracy, help diagnose lesions in anatomically challenging locations, and provide the required standardization with its clinical and academic advantages. we recommend a minimum of 6 photographs of the 6 pelvic zones in the absence of pelvic pathology. in addition, if surgeons dictate according to the zones, comprehensive details will be incorporated into the description report., a comprehensive description of important pelvic structures is frequently missing in operative notes from diagnostic and operative laparoscopy. the anterior cul - de - sac, deep inguinal rings, and the lateral pelvic sidewall peritoneum are the most frequently missed areas. lack of a standardized protocol for photographic documentation is a missed opportunity in providing quality patient care. second, it is comprehensive as it includes all pelvic major structures such as the bladder, uterus, adnexa, and the rectosigmoid colon. it also covers supportive pelvic structures such as the round ligaments, the broad ligament, and the uterosacral ligament. moreover, it describes peritoneal surfaces such as the anterior and the posterior cul - de - sac and the ovarian fossa. in addition, it covers frequently missed areas such as the internal rings and the triangular peritoneal area lateral to the fallopian tube and the infundibulopelvic ligament. lastly, it is easy to follow system whereas the examination could be performed in anteroposterior, then lateral fashion where zone i and ii will be examined first (midline zones). subsequently, lateral zones (right zones iii and iv followed by left zones iii and iv) are to follow. the main limitation of this study was the retrospective use of sources to validate the use of our novel system. in the future, we plan to use operative reports that include photography in order to prospectively describe the six pelvic zones in order to validate this method. by doing this, we propose that more pathology will be diagnosed resulting in improved patient care and communication between surgeons will be improved by extension. | objective. laparoscopic pelvic assessment is often performed in a nonstandardized fashion depending on the surgeon 's discretion. reporting anatomic findings is inconsistent and lesions in atypical locations may be missed. we propose a method for systematic pelvic assessment based on anatomical landmarks. design. retrospective analysis. setting. tertiary care academic medical center. intervention. we applied this system to operative reports of 540 patients who underwent diagnostic or operative laparoscopy for unexplained infertility between 2006 and 2012. the pelvis was divided into 2 midline zones (zone i and ii) and right and left lateral zones (zone iii and iv). all reports were evaluated for the comprehensiveness of description with respect to normal findings or pathology for each zone. results. of 540 surgeries, all reports commented on the uterus, tubes, and ovaries (100%), but only 17% (n = 93, 95% ci : 13.820.2) commented on the dome of the bladder and the anterior cul - de - sac. 24% (n = 130, 95% ci : 20.427.6) commented on the posterior cul - de - sac, and 5% (n = 29, 95% ci : 3.26.8) commented on the pelvic sidewall. overall, 6% (n = 34, 95% ci : 48) reported near complete documentation of the pelvic zones. conclusion. implementation of a systematic approach for laparoscopic pelvic examination will enhance the diagnostic accuracy and provide better communication between care providers. in the absence of pelvic pathology, we recommend a minimum of 6 photographs of the 6 pelvic zones. |
continuous modifications and creative application of new technologies has been the rule. the goal of surgeons has been twofold. first is the reduction of the high recurrence rates seen with traditional open repairs. the second and more humanistic goal has been to reduce the morbidity of the traditional repairs by applying minimally invasive surgical techniques. unfortunately, it is fraught with high complication rates, extended hospital stays, and an unacceptably high recurrence rate that is underestimated by the very surgeons performing the operation. since its introduction to the surgical community 8 years ago, the process of repairing ventral / incisional hernias by laparoscopic techniques has consistently demonstrated a remarkable reduction in recurrence rates, hospital stays, and morbidity. the purpose of this paper is to introduce a technique for laparoscopic repair of ventral / incisional hernias (livh) that is less complex and time consuming than previously reported procedures. like our predecessors, we rely on a wide overlap of a synthetic material intraabdominally to cover the hernia. we have chosen to use composix mesh and composix e / x mesh (davol inc., cranston, ri) for these repairs because the materials utilized in the construction of this mesh allow it to be secured rapidly without the need for transfixion sutures through the abdominal wall. six years of operative experience repairing a wide variety of hernias with this technique has confirmed our position that it is an easily learned technique. more importantly, it grants all the benefits of laparoscopic surgery to patients with ventral / incisional hernias while maintaining recurrence rates that are superior to recurrence rates of open repairs. three steps are required that must be accomplished if the ventral / incisional hernia is to be repaired in a satisfactory fashion : (1) safe abdominal access with proper port geometry, (2) complete lysis of intraabdominal adhesions, and (3) introduction and fixation of an appropriately sized piece of mesh. in many ways, they are made to understand that before any mesh is inserted the adhesions must be removed so that anatomy is clearly seen. a full bowel prep (cathartics and antibiotics) is given the evening before surgery to improve the safety of intraoperative bowel handling and adhesiolysis. we also describe the need for a compressive dressing over the hernia sac in the postoperative period. a 50% reduction in postoperative seromas has been demonstrated with the use of postoperative abdominal wall pressure dressings. in most cases, our patients are asked to continue the compressive dressing until the first postoperative visit 7 to 10 days postoperatively. the procedure is performed with the patient under general anesthesia, and intravenous antibiotics are given in the holding area. in most cases, 1.5 g of ampicillin / sulbactam is given unless patient allergies dictate a change. an orogastric tube is inserted, but a foley catheter is placed only at the discretion of the surgeon. the patient is placed in a low lithotomy position with both arms outstretched on arm boards. typically, a towel roll is placed under the patient 's flank on the primary surgeon 's side. this rolls the patient and target hernia slightly away from the surgeon, which facilitates placement and utilization of the trocars in the lateral abdominal wall. all lines and tubing leave the field at the patient 's shoulders so that the surgeon may move freely from the patient 's left to right sides or even between the legs if safe dissection requires it. initial port strategy is determined by the location of the hernia but may be altered by the degree of adhesiolysis required. in most cases, typically, a single 10- to 12-mm trocar is inserted in a left subcostal position, and 5-mm ports are placed at the transverse umbilical line and just anterior to the anterior superior iliac spine. in all cases, the method of initial port - site insertion is left to the surgeon 's preference and experience. we prefer nonbladed optical ports (optivew, ethicon endo surgery, cincinnati, oh) inserted in an oblique or z - track fashion. our personal experience has shown that when properly inserted they are safe and reduce port - site hernias and complications. often a fourth 5-mm port placed 180 degrees from the previous ports is all that is required. the goals of port placement are to maintain proper triangulation for working, and to eliminate camera angles that result in paradoxical motions on the screen. we feel that the ports need to be far enough away from the hernia so that angled scopes are not required to visualize the defect. this strategy allows us to gain maximum utility from our nonreticulating 5-mm instruments and tacker. over - reliance on angled laparoscopes during a case would suggest that the ports have been inserted too close to the hernia defect. when epigastric, suprapubic, and flank / lumbar hernias are encountered, the port strategy and patient positioning are adjusted. in these special circumstances, we take full advantage of table tilt, patient positioning, and flexible port placement to accomplish the same port strategy goals mentioned above. suprapubic hernias often require a limited preperitoneal dissection to push the bladder down and expose the pubic tubercle and cooper 's ligament for mesh fixation. epigastric hernias typically require a complete takedown of the falciform ligament so that the mesh may extend under the costal margin for added security. we avoid port placement or mesh insertion though the hernia defect itself as any wound complication with such a port could potentially affect or expose the mesh below it. once the ports are appropriately placed, the abdominal cavity is evaluated and any required adhesiolysis is accomplished. all hernia contents are reduced, but no effort to remove the sac is undertaken. if the hernia sac contains bowel, the contents are evaluated to ensure that bowel obstructions will not occur from intraloop adhesions now that they have been mobilized and reduced. blunt dissection is very useful but well vascularized and dense intraabdominal adhesions are lysed with the curved 5-mm lcs harmonic scalpel (ethicon endo surgery, cincinnati, oh). its multi - tasking nature (grasping, dissection, transection, and hemostasis) along with its safety profile relative to monopolar cautery has made it indispensable for many of these cases. when dense intestinal adhesions to prior incisions or mesh make utilization of any energy source hazardous, gentle blunt and sharp dissection with laparoscopic scissors and graspers is used. once the edges of the hernia are clearly visible, the defect size is measured internally by insertion of a small flexible plastic ruler or umbilical tape. external measurements overestimate the true size of the fascial defect when the abdomen is insufflated and are likely to promote insertion of a mesh that is far larger than needed. our goal is to place a prosthesis that extends 3 to 5 cm beyond all edges of the defect. five cm is the preferred overlap but in some circumstances, such as fixation of the mesh to the pubic tubercle or under the costal margin, smaller overlaps are permitted. we have made a transition from using the original composix mesh to the new composix e / x mesh for our repairs. we have found the expanded polytetrafluoroethylene (eptfe) surface on the composix e / x to be more resistant to abrasion during manipulation. the mesh is also thinner than its predecessor, which has advantages during mesh insertion. once the proper size has been determined, it is inserted via the 10- to 12-mm trocar site. we have found that 18 x 23-cm pieces of mesh can be wound around a 5-mm grasper and inserted through the trocar itself. it is important to roll the mesh so that the eptfe surface is on the inside and protected from abrasions from the valves in the trocar. larger pieces of mesh up to and including the 25 x 36-cm piece may be rolled and inserted via the port site after the port is removed. direct insertion via the abdominal wall trocar incision requires the surgeon to roll the mesh so that the marlex surface is on the inside. this prevents unnecessary drag / trauma as the mesh is inserted through the layers of the abdominal wall. the unique incorporation of marlex on the peritoneal side of this particular mesh has allowed us to eliminate the full thickness abdominal wall sutures required for placement and proper fixation of eptfe patches. by eliminating this step, transabdominal transfixion sutures are not necessary for securing or orienting the mesh. the marlex backing on the mesh results in excellent handling characteristics, which simplifies orientation and accurate placement. in most cases, it can be held in place against the abdominal wall with a single 5-mm instrument prior to tack placement. it is recommended that the midline of the mesh be oriented with the vertical axis of the hernia prior to placing the tacks. the first tack should be placed at either the caudal or cephalad edge of this vertical axis. the second tack is then placed 180 degrees from the first after a final check of mesh orientation. drawing a dark line down the vertical axis of the mesh with a sterile marking pen prior to mesh insertion is recommended. when done properly, this technique eliminates the potential for inadequate overlap of the mesh beyond the edges of the hernia. once the mesh is properly positioned with the first 2 tacks, the mesh is rapidly secured with a row of tacks at the periphery of the mesh. these are placed 1 to 2 cm apart to transfix the mesh and to prevent bowel or omentum from slipping under the edge. a second inner row of tacks is placed at the edge of the hernia defect itself with bimanual palpation (hand outside / tacker inside) to ensure proper placement. one hundred consecutive patients have undergone laparoscopic repair of their ventral and incisional hernias with the techniques described above over a 27-month period. most were elective surgeries ; however, 4 cases of incarceration and obstruction were included. three enterotomies (2 small bowel, 1 right colon) occurred in the elective surgical group during blunt or sharp dissection. in each case, no contamination occurred, the bowel was repaired laparoscopically in 2 layers, and mesh was inserted without complication. no injuries occurred related to the use of the harmonic scalpel or the nonbladed optiview trocars. the complexity of the cases has varied widely as has the size of the defects repaired. mesh inserted for repair has varied in size from 10 x 15 cm to 25 x 36 cm. cranston, ri) fused to 1 layer of eptfe was used for the repairs. since the introduction of composix e / x, we no longer use the original mesh. the current mesh has 1 layer of marlex sewn to a thicker layer of eptfe. consequently, the eptfe surface is more resistant to abrasion during placement, and larger pieces can be inserted via the trocar. infections have occurred. to date, no postoperative bowel obstructions or readmissions for pain control or ileus have been necessary. one morbidly obese patient required urgent exploration and drainage of a large subcutaneous and intramuscular flank hematoma resulting from the use of a bladed 5-mm trocar. in the last 6 years, we have gained a great deal of practical experience with livh repair by caring for our own patients and through teaching other surgeons. we have learned that the most important details relate to (1) proper port geometry, (2) safe adhesiolysis, and (3) inserting an appropriately sized mesh to maintain adequate overlap beyond the margins of the hernia. the initial repair involved multiple widely spaced hernias covered with a single large 25 x 36-cm prosthesis. on reexplo - ration, it was obvious that the original repair was still intact and well incorporated. the recurrence was small but had occurred at the edge of the mesh closest to the working ports. inadequate prosthetic overlap on the near side was the likely cause of this preventable recurrence. incisional hernias remain a problem in many surgical specialties despite improvements in materials and adoption of methods like the mass closure technique. aside from obesity and wound infection it has been estimated that close to 10% of incisions will develop hernias. a review of 655 patients whose incisions were closed with a running monofilament utilizing a mass suturing technique revealed an incisional herniation rate of 14.2% by 2 years, with 8.4% presenting in the first year. in general, 60% to 90% of incisional hernias are observed in the first 2 to 3 years after major laparotomies. unfortunately, the recurrence rates associated with traditional open repairs are unacceptably high. a modern trial of 200 patients undergoing suture or mesh repair of a primary hernia or a first recurrence reported a 3-year recurrence rate for primary hernia repair of 43% in the suture group and 24% in the mesh group. these recurrence rates climbed to 58% when suture alone was used to close recurrent hernias. they noted that the size of the hernia did not affect the rate of recurrence and that mesh repair (retrofascial preperitoneal polypropylene) was superior to suture repair alone. the traditional vertical mayo or vest - over - pants repair has been shown to have a 48% recurrence rate in the first 3 years when used to repair midline incisional hernias. historically, primary tissue repairs have been associated with a 30% to 50% recurrence rate. although the adoption of tension - free repair techniques with mesh prostheses has dropped the recurrence rates to the 10% to 20% range, it is still unacceptably high. one series of 250 hernias repaired over 14 years yielded a wound complication rate of 34%. the complications being reported were infection, seroma formation, hematoma, and fistula formation. those patients in whom mesh was used during their repair suffered a wound complication rate of 49% and an infection rate of 18%. in 1987, stoppa reported an infection rate of 18.5% in his series of 230 retrorectus mesh repairs. more recently the use of prosthetic materials in open ventral hernia repairs reduces the risk of recurrent hernias but carries the risk of increasing wound infections. it is not unreasonable to presume that the large amount of dissection and tissue undermining / devascularization required to accomplish these repairs has contributed to the infection rate. the pain associated with the dissection and mesh fixation certainly plays a role in the need to admit many of these patients to the hospital postoperatively. the improved results have been maintained despite variations in technique and prosthetics from study to study (table 1). the common denominator in all successful livh repairs is the intraperitoneal placement of the prosthesis. in general, the only things that have changed from one study to the next are the types of prostheses being inserted, the degree of hernia overlap, and the manner of fixation. we have previously demonstrated that more than 95% of these patients can be discharged from the recovery room regardless of the size of the defect. others have demonstrated a significant decrease in facility costs when comparing open and laparoscopic repairs as 90% of their laparoscopic hernia repairs did not require inpatient care. shorter hospital stays and less severe postoperative complications in laparoscopic repairs contributed to statistically significant lower costs relative to open repairs in the same study. a group of surgeons cut their hernia recurrence rate by nearly 50% when they switched from open placement of eptfe to laparoscopic placement. they declared the decrease in postoperative pain, complications, and lowered recurrence rate to be a great benefit to our practice. it is difficult to accurately catalogue the various laparoscopic techniques that have been tried in an effort to reduce recurrence rates and complications associated with traditional anterior repairs. they are often simply a reflection of the technology available at that particular time and the skill of the surgeon involved. some aspects of the repairs such as patient preparation, trocar placement, and lysis of adhesions change very little from report to report.. early attempts at laparoscopic ventral hernia repair were often a reflection of what was being taught for inguinal hernia repair. preperitoneal dissections were made and polypropylene mesh was placed over the defect and stapled to the abdominal wall. some have referred to it as being virtually prohibitive. as a result, methods for complete intraabdominal mesh placement have been evaluated. although surgeons were familiar with polypropylene mesh from open hernia repairs, many were worried about the complications that could develop from a complete intraabdominal placement of this material. in some techniques, the omentum is used to act as a barrier between the polypropylene and the bowel. in fact, many authors began to use eptfe in their repairs despite its much higher cost. they felt that the possible reduction in both intraabdominal adhesions and the risk of bowel fistualization justified the cost of the eptfe. despite these concerns, some surgeons have produced large series of patients repaired with polypropylene that have excellent results and no evidence of complications related to bowel adherence to the mesh. despite advantages in terms of intraabdominal adhesion formation, eptfe presented its own unique set of challenges for surgeons. very experienced laparoscopic surgeons have described manipulation of this mesh as a cumbersome procedure and difficult to work with laparoscopically because of its lack of memory and the fact that it is opaque. surgeons also began to realize that hernia staplers were inadequate to transfix this material to the abdominal wall and began looking for alternatives. in 1996, none of the currently available articulating endoscopic staplers consistently and adequately secured a 1-mm ptfe patch to a depth beyond the peritoneum. he and others began to increase the length of their overlap to 3 cm or greater and to utilize full thickness transabdominal stay sutures to secure the mesh. these measures also serve to compensate for the relative lack of fibrous ingrowth into the ptfe relative to polypropylene. ptfe repairs rely heavily on the integrity of the suture attachment of the prosthesis to the fascia. failure to place these full - thickness sutures in ptfe repairs results in higher recurrence rates. when eptfe is properly sized and transfixed, excellent results can be achieved with laparoscopic ventral hernia repairs (table 1). unfortunately, some of the mesh - specific needs of eptfe can make these repairs difficult for surgeons who do not have a great deal of experience with the technique. in some cases, the proper placement and orientation of the sutures can add up to 50% to the time needed for repair. these sutures represent a potential portal for contamination of the mesh with skin flora, risking infection and possible mesh removal. reports have also been made of long - term pain associated with the sutures, and in some cases second procedures have been necessary to remove the offending suture. we have found composix mesh and now composix e / x mesh to be excellent prostheses for the laparoscopic repair of ventral and incisional hernias. their eptfe surface protects the visceral organs from significant adhesions while the polypropylene surface provides a dependable template for rapid fibroblastic ingrowth. additionally, the polypropylene layer gives the mesh enough memory to make it easy to manipulate both small and large pieces in a laparoscopic field. we feel the technique we have described for livh repair is one that can be easily replicated by most general surgeons without the need for advanced training in laparoscopic surgery. surgical technique for repair of hernias is as old as the profession itself. over time, the accepted gold standards for repair of specific subsets of hernias have been challenged and often replaced. standard techniques is often the result of technical innovation and the availability of novel materials. until the recurrence rate reaches zero and pain is eliminated from the process of hernia repair, we will continue to strive to do better. the contemporary results of open incisional and ventral hernia repair are unsatisfactory because of high recurrence rates and morbidity levels. the minimally invasive approach to repairing even complex ventral and incisional hernias is gaining rapid acceptance across the country due to its lower recurrence rate, reduced complication rates, and patient satisfaction. the location for laparoscopic repair of these hernias is quickly moving from the large academic centers to community hospitals as surgeons educate themselves on newer, less awkward techniques for mesh insertion and fixation. a gold standard for laparoscopic ventral and incisional hernia is difficult to develop when the products being used change so rapidly. to our advantage, newer - generation products (ie, nonbladed optical trocars, ultrasonic shears, smaller tackers, and improved mesh composites) are making these repairs safer for patients and easier to learn for surgeons who do not have the benefit of advanced laparoscopic training consequently, surgeons must keep an open mind about all of the products and techniques being offered to facilitate the repair now and in the future. | background and objectives : the contemporary results of open incisional and ventral hernia repair are unsatisfactory because of high recurrence rates and morbidity levels. laparoscopic repair of ventral and incisional hernias (livh) can be accomplished in a simple, reproducible manner while dramatically lowering recurrence rates and morbidity.methods:one hundred consecutive patents underwent laparoscopic repair of their ventral and incisional hernias over a 27-month period. composix mesh and composix e / x mesh (davol inc., cranston, ri) were utilized for the repairs. transfixion sutures were not used.results:all repairs were completed laparoscopically. no conversions to open techniques were necessary. no postoperative infections have been observed. one recurrent hernia was identified and subsequently repaired with the same technique.conclusions:livh can be accomplished with a dramatic reduction in recurrence rates and morbidity. the technique for this repair is still in a state of evolution. the construction and handling characteristics of this particular type of mesh have allowed us to eliminate transfixion sutures and to simplify the repair technique while maintaining a very low recurrence rate. |
the fetus relies exclusively on maternal thyroid hormone (th) in early pregnancy for growth, neurodevelopment, and the regulation of metabolic processes [2, 3 ]. dysregulation in glucose and lipid metabolism has been associated with many metabolic syndromes including gestational diabetes mellitus (gdm) and th imbalance is a contributing factor to these diseases [4, 5 ]. the global prevalence of gdm and obesity is rising in the obstetric population and their effects on maternal and fetal outcomes are well documented [68 ]. caloric intake and personal lifestyle are strongly associated with obesity and metabolic syndrome ; however, there is a growing concern that a subgroup of endocrine disrupting chemicals (edcs) known to specifically alter th regulation (th - edcs) may contribute to the prevalence of metabolic syndromes by altering signalling pathways involved in glucose and lipid homeostasis during pregnancy. the risk of exposure to th - edcs is rising with exposure to multiple th - edcs more common than exposure to single chemicals. several studies have reported the effects of exposure to individual edcs and this has been reviewed elsewhere. this review discusses the effects of combinations of th endocrine disruptors on the regulation of glucose and lipid metabolism during pregnancy. for this narrative review conducted between 2014 and 2015, the pubmed (us national library of medicine, national institutes of health) and google scholar databases were interrogated with the following key words and phrases : th endocrine disruptors and pregnancy / early or late pregnancy ; prenatal effect of th endocrine disruptors on glucose / lipid metabolism ; mechanism of action of th endocrine disruptors ; placental transfer / biotransformation of th endocrine disruptors. all study types including randomized controlled trials, case - control, human, and animal studies were considered and results are restricted to english only. the articles were grouped according to the effects of the th - edcs on related endpoints. the ths thyroxine (t4) and triiodothyronine (t3) are synthesized and secreted by the thyroid gland. thyroid epithelial cells synthesize thyroglobulin, which provides tyrosine residues that are iodinated to iodotyrosine by thyroid peroxidase (tpo) to form t4 and t3 (20% of total t3 is made by the thyroid gland). maintenance of blood th levels occurs through a hypothalamic - pituitary - thyroid axis feedback mechanism. basically, tsh - releasing hormone (trh) in the hypothalamus stimulates thyroid stimulating hormone (tsh) secretion from the anterior pituitary, which in turn initiates th synthesis and release from the thyroid gland. th also acts at the transcriptional level to suppress the synthesis of trh and tsh (figure 1). ths are highly lipophilic and are secreted into the blood stream where they are bound by the th distribution proteins thyroxine binding globulin (tbg), transthyretin (ttr), or albumin. th cellular uptake is regulated by cell specific expression of th transporters including organic anion - transporting polypeptides (oatps), large neutral amino acid transporters (lats), and monocarboxylate transporters 8 and 10 (mct8, mct10) (figure 2). once inside the cell, the deiodinase enzymes (type 1 (d1), type 2 (d2), and type 3 (d3)) regulate the conversion of t4 to either active t3 (d1 and d2) or inactive reverse t3 (rt3) (d1 and d3) by the removal of a specific iodine atom. metabolizing enzymes such as uridine 5-diphospho - glucuronosyltransferases (udp - gt) and sulfotransferases also regulate th bioavailability by rendering ths more water soluble and more easily excreted. ths converted to t3 in the cell can either activate or repress gene transcription by binding to nuclear th receptors (trs) which are dna - binding transcription factors that bind specific thyroid hormone response elements (tres) in the regulatory region of target genes. trs may bind tres as homodimers but also commonly bind as heterodimers with other nuclear receptors such as the retinoid x receptor (rxr). a person is described as euthyroid when the thyroid gland is functioning normally resulting in normal steady state levels of serum ths and tsh. hypothyroidism results when the thyroid gland does not produce adequate amounts of th resulting in low serum th levels and elevated tsh. hyperthyroidism results from the thyroid gland producing too much th resulting in high th and suppressed tsh in the serum. both hypo- and hyperthyroidism commonly result from autoimmune disease attack of tpo (hypo- and hyper-), thyroglobulin (hypo-), tsh receptor antibodies, and thyroid stimulating immunoglobulin (hyper-). when serum th levels fall within the normal range but tsh levels are not normal (autoantibodies are also often present), this signifies the early stages of thyroid disease and is described as a subclinical state. th disrupting chemicals would clearly have different effects on individuals with different types of thyroid disorder. the placenta secretes a glycoprotein hormone called human chorionic gonadotropin (hcg), which possesses structural similarities to tsh and acts as a weak tsh receptor agonist. due to elevated levels of estrogen and hcg in pregnancy, a marginal increase in the basal tsh and serum tbg as well as alterations in th levels and peripheral metabolism of maternal th is believed to alter the thyroid hormone system. the maternal thyroid gland accommodates the demands of pregnancy and a growing fetus by increasing hormonal output. the human fetal thyroid can secrete th from 16 weeks of gestation, whereas prior to this the fetus is dependent on transplacental supply of maternal t4. in the first trimester, there is a parallel increase in total t4 concentration in the maternal and fetal compartments. this was attributed to high fetal d3 and sulfotransferase activities and confirmed by reports of high d3 activities in fetal hepatic and placental tissues [44, 45 ]. in addition, due to lower levels of binding proteins such as ttr in the human fetus, a higher proportion of maternally transferred t4 exists as free t4 in the coelomic fluid cavities. ttr is produced and secreted by the human placenta and binds t4 with high affinity and may transport maternal t4 to the fetus. glucose metabolism is altered during pregnancy to guarantee adequate delivery of nutrients to the growing placenta and fetus. in pregnancy, maternofetal glucose transport is largely dependent on the expression of one or more member of the facilitative family of glucose transporters (gluts) in the placenta. increased fat mass in overweight and obesity is associated with prepregnancy insulin resistance, which is compounded by insulin resistance of pregnancy [47, 48 ]. in late pregnancy, insulin resistance is associated with hepatic gluconeogenesis and decreased utilization of glucose by peripheral tissues which contribute to meeting the increasing placental and fetal energy demands [49, 50 ]. with higher prepregnancy insulin resistance, for example, in obesity, the additional pregnancy - induced insulin resistance can not always be compensated for by increased insulin secretion resulting in overt hyperglycemia. gestational diabetes mellitus (gdm) is hyperglycemia first detected during pregnancy and may affect up to 17.8% of pregnancies. the etiology of gdm has been attributed to genetic predisposition, insulin resistance, and altered beta cell function [5254 ]. th affects many aspects of glucose metabolism : circulating insulin levels and counter - regulatory hormones ; hepatic gluconeogenesis and glycogenolysis ; and intestinal glucose uptake. th regulates the transcription of several genes involved in glucose metabolism. at the tissue level, th actions on glucose metabolism are regulated by transmembrane transporters, ligand - dependent th receptors, and deiodinases. deranged th levels in pregnancy are associated with adverse maternal and fetal metabolic outcomes [4, 56, 57 ]. excess th increases basal metabolic rate with a concomitant increase in the demand for glucose. in response to excess th, pancreatic cells continuously secrete insulin even in the absence of stimulatory glucose concentrations. untreated hyperthyroidism, although uncommon in pregnant women, may lead to restricted fetal growth, stillbirth, or preterm delivery due to reduced availability of nutrients to the fetus [60, 61 ]. furthermore, in healthy pregnant women with normal prepregnancy thyroid function, lower free t4 concentrations and a higher conversion of free t4 to free t3 are positively correlated with elevated glucose levels after oral glucose load as well as higher fasting insulin when adjusted for bmi. this suggests that variations in th levels within euthyroid concentrations also impact glucose metabolism in pregnancy. in contrast, evidence of decreased glucose production and utilization has been observed in overt and subclinical hypothyroidism. th replacement therapy in patients with subclinical hypothyroidism resulted in a partial reversal to the euthyroid state with significant reductions in glucose - stimulated - insulin secretion (gsis). this implies that poorly managed hypothyroidism could result in high nutrient availability for the fetus, leading to large gestational age infants predisposed to obesity later in life. animal studies of maternal hypothyroidism have reported impaired brain development perhaps due to altered expression of glut protein isoforms in the placenta and fetal brain. the dynamics of lipid metabolism during pregnancy vary with gestational age : accumulation of fat in early pregnancy (anabolic phase), shift to increased insulin resistance, decreased adipose tissue lipoprotein lipase (lpl) activity, and increased lipolysis in late pregnancy (catabolic phase). the rise in maternal insulin resistance allows more glucose to be transported to the fetus in late pregnancy while the mother utilizes lipids as her main energy source. the placental transport of fatty acids to the growing fetus is mediated by lipoprotein receptors, lipid carrier proteins, and actions of lipase enzymes. the exact mechanisms of placental lipid transport are however still unclear. maternal lipid metabolism in normal and complicated pregnancies has recently been reviewed. in women with gdm, the higher levels of insulin resistance are associated with increased adipose tissue lipolysis and higher free fatty acids and triglycerides in maternal serum, increasing fetal nutrient availability resulting in fetal overgrowth. the expression of th receptor isoforms is an important determinant of its actions on lipid metabolism. the overall effects of th on lipid metabolism are the sum of its actions primarily in the liver and adipose tissue. in the liver, th increases hepatic cholesterol uptake and synthesis mainly not only by inducing the transcription of the ldl - receptor but also by reducing apolipoproteins b48 and b100, which is associated with increased hepatic triglyceride production. the higher th levels in pregnancy may contribute to the increased lipid levels observed in the maternal circulation. altered th levels have been associated with a less favorable metabolic phenotype in pregnant women with normal thyroid function prepregnancy [56, 72 ]. hyperthyroidism is characterised by an increased turnover of ldl cholesterol resulting in decreased plasma lipid levels. in contrast, hypothyroidism in late pregnancy has been associated with enhanced cholesterol levels and decreased triglycerides and hdl - cholesterol levels in rats. because of the importance of th in regulating glucose and lipid metabolism in pregnancy and thereby the growth of the baby, substances that affect th may have large effects on the health and development of mother and infant (table 1). exogenous substances capable of interfering with the structure or function of the endocrine system are known as endocrine disrupting chemicals (edcs). as with other hormones, th is a target of edcs and many studies have identified chemicals that alter th homeostasis. th - edcs may be clustered into two main groups based on their biodegradability and bioaccumulation in the environment. nonpersistent organic chemicals (n - pocs) are widespread in the environment but are nonlipophilic and do not bioaccumulate. persistent organic chemicals (pocs) are highly stable lipophilic compounds that bind to adipose tissue in living organisms and bioaccumulate up the food chain. n - pocs and some pocs are rapidly metabolized by enzymes and eliminated from the body. organochlorine (oc) and organophosphate (op) pesticides are highly lipophilic ; ops are unstable and therefore more readily metabolized than ocs which have been found to accumulate in adipose tissue. th - edcs are ubiquitous and originate from chemical additives used as flame retardants ; synthetic plasticizers and solvents used in food packaging, polyvinyl chloride tubing, medical equipments, pesticides, toys, personal products, adhesives, powder paints, and dental sealants ; antimicrobial compounds used in household detergents ; toxic by - products of combustion processes ; insulating materials for electrical equipment such as transformers and capacitors, heat transfer systems, hydraulic fluids, and lubricants ; airbag inflation systems, fireworks, nitrate fertilizers, matches, and oxidants in propelling rockets and missiles ; and synthetic and naturally predominant compounds in soy rich foods [9, 16, 76 ]. humans come in contact with these chemicals through ingestion, inhalation, dermal exposure to contaminated substances, and intravenous and parenteral absorption from medical devices containing phthalates. th - edcs interfere with th and tsh signalling through many pathways in many different species : altering deiodinase activity [25, 28, 77 ], inhibiting th excretion or metabolism [26, 29 ], blocking iodine uptake by thyroid cells, competitively binding the thyroid transport protein ttr, the inhibition of human tpo [36, 37 ], and acting as an antagonist of complexes from the thyroid hormone responsive elements (tres) [19, 78 ]. certain th - edcs such as brominated flame retardants, hydroxylated polychlorinated biphenyls (pcbs) metabolites, and dioxin (pcdd) share structural similarities with th and bind with the high affinity th transport protein ttr, consequently inhibiting t4-ttr binding. in serum samples from polar bears, t4 binding sites on ttr in pregnant rats, reduced fetal plasma and brain total t4 levels in response to prenatal exposure to hydroxylated pcbs were hypothesized to be due to the binding to ttr. many th - edcs not only affect th but also interfere with the actions of other hormones acting through nuclear receptors such as sex hormones (estrogen, progesterone, and androgen) or by interacting with their respective nuclear receptors (er, pr, and ar). in vitro toxicology studies of non - dioxin - like pcbs on humans revealed that pcb related compounds (congener) 168 and 125 completely inhibited t4-ttr binding because of their structural similarity to t4, but they also had very high androgen - inhibitory potencies. on the other hand, pcb-104 had high ar - antagonistic potencies and was the most effective congener with estrogenic properties of all congeners studied. exposures to th - edcs in pregnancy have been associated with alterations in th regulation and adverse pregnancy outcomes (table 2). the effects of th - edcs on the levels of th or its receptor will affect downstream signalling including metabolic pathways. tr - beta is the main tr isoform in both liver and adipose tissue and bpa exposure could therefore reduce both lipogenesis and lipolysis reducing lipid availability in the circulation. furthermore, the interactions between the tr and other nuclear receptors such as the rxr and fxr receptors further complicate the effects of edcs since many th - edcs such as bpa independently affect other nuclear receptors which also affect lipid and glucose metabolism. it may be possible to predict the effects of combinations of edcs based on their individual th signalling targets. a summary of th signalling targets of different th - edcs is presented in table 3. in modern society, most individuals will have been exposed to mixtures of edcs rather than single edc. therefore, the study of the effects of combined chemicals is critical to reach meaningful conclusions on the plausible role of these chemicals on human health. effects of edcs can either be additive, synergistic, or antagonistic since chemicals may interact with one another to modify the nature of the toxic effect. for instance, human studies revealed significant associations between increased levels of pcbs, dichlorodiphenyldichloroethylene (dde), and hexachlorobenzene (hcb) with adverse thyroid volumes and multiple metabolic disorders, especially in older subjects. complex mixtures may therefore result in more or additional deleterious effects on metabolism (figure 2). prenatal exposure to edcs continues to pose serious health risks to developing fetus and children as evidence of adverse effect on birth outcomes, childhood obesity, and intellectual disability are increasing [4, 84, 85 ]. more importantly, because organogenesis begins at the time when the fetus is solely dependent on maternal th supply, early life exposure to th - edcs may lead to adverse short or long term health outcomes due to fetal reprogramming [86, 87 ]. the placental barrier is not impervious to th - edcs as many of them have been measured in human fetal cord blood, neonatal meconium, rat fetal serum, and human amniotic fluid. th - edcs are able to traverse the placental barrier by diffusion or via an active transporter (such as oatps) either as pure or as biologically transformed chemicals (through conjugation of chemicals by placental metabolizing enzymes such as sulfotransferases (sults) and udp - glucuronosyl transferases (ugts)) (figure 3). biotransformation of chemicals by metabolizing enzymes makes the chemicals fit for excretion and may result in inactivation or increased toxicity. the biotransformed compounds can be extruded into the maternal circulation for excretion via placental transporters such as multidrug resistant - associated proteins. expression of metabolizing enzymes and transporters has been shown to vary with gestational age in human and animal placentas [91, 92 ]. the majority of the enzymes and transporters are expressed throughout human pregnancy with decreasing expression observed as pregnancy advances (e.g., ugt1 [93, 94 ] : p - glycoprotein (p - gp)) whereas others are expressed during late pregnancy and continued during postnatal period. the decline in the expression of transporters with gestational age is further corroborated by the increased rate of placental transfer and amount of p - gp substrates during late pregnancy compared to early pregnancy. some th - edc chemicals may alter other metabolic pathways thereby modulating the effect of metabolizing enzymes on endogenous substrates like steroid hormones or other xenobiotics. ex vivo studies on human term placentas demonstrated that environmentally relevant levels of bpa in mothers freely diffuse across the placenta in an unconjugated form suggesting that sults and ugts play a minor role in the transplacental transport of bpa. this was attributed to the low expression of placental enzymes involved in metabolizing the chemicals towards the end of pregnancy. a similar study on term human placentas revealed that genistein, a naturally occurring phytoestrogen was able to traverse the placental barrier although only a small proportion was biotransformed by metabolizing enzymes in the placenta. ugt activity has been shown to be higher in human first trimester placenta compared to term placentas and to decrease as pregnancy advances in humans. this implies that the fetus is more protected from the toxic effect of th - edcs by ugt enzymes in early pregnancy than in late pregnancy, which might be a consequence of the vulnerable detoxification system in the infant in early pregnancy. this is different from animal models ; here bpa actively traversed the rat term placenta predominantly in its conjugated form and was deconjugated by -glucuronidase enzymes in the rat fetus. in addition to the low ugt activity observed in the rat fetus, high levels of oatp4a1 and mrp1 transporters were expressed at the maternal and fetal interfaces of the placenta, respectively, suggesting that conjugated bpa is transferred from mothers by oatp4a1 to the growing fetus by mrp1 leading to toxic effects of chemicals on the fetus. prenatal exposures to mixtures of th - edcs have revealed diverse ways by which chemicals alter th homeostasis. perinatal exposures to low doses of bpa increase abdominal adipocyte tissue mass and correlate with hyperlipidemia in a dose - response manner in mice. however, exposures to low concentration of bpa had no adipogenic effect on murine mesenchymal stem cells in vitro. although the nonmonotonic effect of bpa, characterized by high responses at low and high exposure levels, is well known, it is worth mentioning that a much lower bpa concentration was administered to the pregnant mice than the mesenchymal stem cells. this discrepancy may be a result of the insulin resistant state in pregnancy, which is related to decreased adipogenesis. in normal pregnancy, placenta - derived hormones induce a state of insulin resistance with the aim of maintaining adequate energy supplies to the placenta and developing fetus. insulin is known to promote lipogenesis in adipose tissue while suppressing lipolysis therefore an insulin resistant state will result in reduced lipogenesis while at the same time favoring lipolysis. a study of individual chemical effects on adipogenesis in animal models demonstrated an association between high doses of bpa and decreased adipocyte and lipid levels whereas diethylhexylphthalate (dehp) and tributyltin (tbt) were linked to enhanced adipogenesis. however, when studied together, the negative effect of bpa on adipogenesis was outweighed by the positive effect of dehp and tbt on the proliferation of adipocytes at high concentrations with increased adipogenesis even though the effect was not as profound as observed with individual dehp and tbt chemicals. this suggests that the effect of chemical mixtures can not be predicted from outcomes from individual chemicals due to varied mechanisms of action. in this case, bpa signals through the regulation of estrogen even though studies of bpa itself have shown that bpa can alter th regulation. on the other hand, dehp and tbt both activate the peroxisome proliferator - activated receptors gamma (ppar) signalling pathway. altered th levels and postnatal thyroid function have been reported in preterm infants. epidemiological studies have revealed inverse or positive associations between maternal urinary bpa and mono-2-ethyl-5-hydroxyhexyl phthalate (mehhp) with th levels in adults and adolescents, respectively [105, 106 ]. also increases in bpa and mehhp are associated with significant reductions in gestation in male offspring specific pregnancies in one but not in another study. higher maternal prepregnancy bmi and higher gestational weight gain are correlated with higher birth weight and fat mass at birth and increased bmi in young and adult offspring [109, 110 ]. in addition, maternal preexisting diabetes and gestational diabetes have been associated with increased birth weight and development of later childhood obesity. in a monotonic relationship, an increase in the dose of a chemical is attended by a corresponding increase in the effect of the chemical on the observed endpoint. likewise, there is a corresponding decline in the effect with decreasing the doses of the chemicals. in male neonates, monotonic relationships were observed between phenols and birth weights ; however, a u - shaped nonmonotonic association existed between phthalate metabolites and birth weights. fetal exposures to phthalates and dichlorodiphenyldichloroethylene (dde) have been associated with increased bmi and head circumference (hc) during the first year after birth in a nonmonotonic manner. animal studies revealed that exposures to a low dose of mehp during pregnancy significantly increased body weight and fat pads of male offspring at 60 days after delivery, as well as serum cholesterol, triacylglycerol (tag), and glucose levels in mice. this demonstrates that low - dose effects can not be predicted from outcomes at higher doses. the specific effect on male offspring may be due to the interference of mehp with male hormones as human epidemiological studies have revealed an association between mehp and decreased steroid hormones in adult males. sex hormones have been linked to obesity with positive relationship observed between androgen levels and bmi in females while a negative relationship exists between levels of androgen and small waist circumference in males. in breast cancer cells, hence, it can be speculated that, at low mehp levels, the ratio of androgen to estrogen is high, therefore resulting in the observed obesity phenotype in male offspring. however, in female fetuses with higher estrogen to androgen levels, low level mehp exposure would lead to estrogen inhibition of ppar activity and lipid metabolism. perinatal exposure to polychlorinated biphenyls (pcbs) and dioxins and their effects on puberty have been reviewed. exposure to pcb / dioxin has been linked to increased obesity, disruption of sex hormone signalling, retardation in the growth, and development of sex organs in males and breasts in girls. pcb / dioxin exert their role on puberty by interacting with the ahr which interferes with hormonal systems. prenatal exposure to a combination of pcbs and dichlorodiphenyldichloroethylene (dde) may predispose female offspring to obesity in overweight mothers. exposure to dde alone in infants of normal - weight women leads to more weight gain in the first 6 months postnatally and high bmi in early childhood. polychlorinated dibenzo - p - dioxins (pcdds) and polychlorinated dibenzofurans (pcdfs) were found to significantly affect birth weight among male infants but not among female infants. however, dietary dioxin and pcb intake was not associated with the risk for small - for - gestational age neonates. this implies that some chemicals may have differential gender effects by interacting with some gender specific hormone receptors and signalling pathways such as estrogen and androgens. many studies have associated exposures of persistent organic pollutants with th dysregulation [27, 28, 30 ]. the effects of some chemicals may be impacted by confounding factors such as maternal demographic and perinatal factors. maternal gestational weight gain (gwg) has been implicated to influence the overall burden of persistent organic pollutants (pops) in neonates. in the anabolic phase of pregnancy, mothers use less of their stored fats, which would reduce the proportion of pops that get liberated from fat stores. on the contrary, late pregnancy is marked by increased lipolysis, which may contribute to the increased release of compounds trapped in stored lipids, leading to an even higher exposure in the fetus. it could thus be that the level of exposure to pops varies over the course of pregnancy. a negative association was recorded between pbde congeners and birth weight ; however, when adjusted for maternal weight gain, these findings were no longer statistically significant. also, no association was observed between pbdes and birth length, head circumference, or gestational duration. exposure to pcbs and pops during pregnancy has been linked to adverse effects on fetal growth [127129 ], increased birth weight, and shortened length of gestation in humans. prenatal exposures demonstrated a link between dde and hcb with early postnatal growth but not with pcb. it is crucial that confounders be identified in studies to rule out false positive results and to determine their role in maternal and fetal outcomes. the role of th - edcs on metabolic risk factors such as insulin resistance, glucose tolerance, and triglycerides and cholesterol levels has only recently become the subject of research. early studies suggest that th - edcs may act via a variety of mechanisms and that studies using high doses of these chemicals are probably not good predictors of effects at low doses. additionally, low doses of combinations of chemicals, which would be a better reflection of the current situation, have shown conflicting results on the endpoints investigated. variations in the effects of th - edcs on fetal growth have been reported with respect to gender and gestational age. in order to provide better prenatal care and improved health outcomes, it is important that studies be carried out to evaluate the effects of th - edcs on lipid levels, insulin sensitivity, and glucose tolerance as they are significant underlying factors in the development of metabolic syndrome. | background. thyroid hormones (ths) are essential for normal human fetal development and play a major role in the regulation of glucose and lipid metabolism. delivery of th to target tissues is dependent on processes including th synthesis, transport, and metabolism. thyroid hormone endocrine disruptors (th - edcs) are chemical substances that interfere with these processes, potentially leading to adverse pregnancy outcomes. objectives. this review focuses on the effects of prenatal exposures to combinations of th - edcs on fetal and neonatal glucose and lipid metabolism and also discusses the various mechanisms by which th - edcs interfere with other hormonal pathways. methods. we conducted a comprehensive narrative review on the effects of th - edcs with particular emphasis on exposure during pregnancy. discussion. th imbalance has been linked to many metabolic processes and the effects of th imbalance are particularly pronounced in early fetal development due to fetal dependence on maternal th for proper growth and development. the pervasive presence of edcs in the environment results in ubiquitous exposure to either single or mixtures of edcs with deleterious effects on metabolism. conclusions. further evaluation of combined effects of th - edcs on fetal metabolic endpoints could improve advice provided to expectant mothers. |
diabetes is rapidly increasing in prevalence worldwide and surgery in patients with diabetic foot is becoming more common. foot complications are a major cause of admissions in diabetic patients, and comprise a disproportionately high number of hospital days because of multiple surgical procedures and prolonged length of stay in hospital. foot disorders such as ulceration, infection and gangrene are the most common, complex and costly sequelae of diabetes mellitus.[46 ] the optimal therapy for diabetic foot ulcers remains ill - defined. saline - moistened gauze has been the standard method ; however, it has been difficult to continuously maintain a moist wound environment with these dressings. this has led to the development of various hydrocolloid wound gels, which provided more consistent moisture retention. refinements in topical ointments have resulted in the addition of various pharmacological agents including growth factors and enzymatic debridement compounds. hyperbaric oxygen therapy and culture skin substitutes are other wound therapies that have been advocated. all these therapies are associated with significant expense and are being utilized in some situations without sufficient scientific evidence demonstrating their efficacy. therefore, the search for an efficacious, convenient and cost - effective therapy continues. negative pressure wound therapy (npwt) is a newer noninvasive adjunctive therapy system that uses controlled negative pressure using vacuum - assisted closure device (vac) to help promote wound healing by removing fluid from open wounds through a sealed dressing and tubing which is connected to a collection container. the use of sub - atmospheric pressure dressings, available commercially as a vac device, has been shown to be an effective way to accelerate healing of various wounds.[710 ] till today, very limited data is available on the role of negative pressure dressing in healing of diabetic foot ulcers. therefore, we endeavor to put forward a study to evaluate the role of negative pressure dressing in healing of diabetic foot ulcers using vac device. the present study was done on 30 patients at dayanand medical college and hospital, ludhiana. control group (b) : received twice daily dressing changes with saline - moistened gauze. age group 20 - 75 years.ulcer area ranging between 50 cm and 200cm.diagnosis of diabetes mellitus made by american diabetes association criteria. age 75 years.an obvious septicemia.osteomyelitis.wounds resulting from venous insufficiency.malignant disease in a wound.patients being treated with corticosteroids, immunosuppressive drugs or chemotherapy.any other serious pre - existing cardiovascular, pulmonary and immunological disease. wounds of the subjects included in the study underwent initial sharp debridement to remove necrotic tissue and slough as far as possible. after the debridement, foam - based dressing was done over the wounds of the study group under all aseptic conditions. an evacuation tube embedded in the foam was connected to a fluid collection canister contained within a portable vacuum / suction machine [figures 1 and 2 ]. subatmospheric (negative) pressure was applied within a range of 50 mmhg to 125 mmhg intermittently three times a day. weekly cultures were taken from the floor of the ulcers to assess for the bacterial flora. standard antibiotic regimes were administered to all the patients which consisted broad spectrum antibiotics initially and later according to the culture sensitivity report. ulcers were treated until the wound got closed surgically or spontaneously, or until completion of the 56-days (8 weeks) assessment whichever was earlier. showing application of negative pressure wound therapy showing machine used to create negative pressure complete healing was defined as 100% wound closure with re - epithelialization or scab with no wound drainage present and no dressing required. at the end of the study period patients were categorized as : complete responders : complete healing of lower limb ulcers.partial responders : 50% or greater reduction in product of the two longest perpendicular diameters from baseline.noncomplete responders : less than 50% reduction in the product of the two longest perpendicular diameters from baseline.nonresponders : no reduction in ulcer or increase in ulcer area over base line. partial responders : 50% or greater reduction in product of the two longest perpendicular diameters from baseline. noncomplete responders : less than 50% reduction in the product of the two longest perpendicular diameters from baseline. the observations were noted and all results were tabulated and analyzed by using student t - test for age, fasting blood sugar and percentage change in wound size from 1st to 8th week. the appearance of granulation tissue and the primary study end point were tested for significance by applying test. the analysis for time status of wound age group 20 - 75 years.ulcer area ranging between 50 cm and 200cm.diagnosis of diabetes mellitus made by american diabetes association criteria. age 75 years.an obvious septicemia.osteomyelitis.wounds resulting from venous insufficiency.malignant disease in a wound.patients being treated with corticosteroids, immunosuppressive drugs or chemotherapy.any other serious pre - existing cardiovascular, pulmonary and immunological disease. patients being treated with corticosteroids, immunosuppressive drugs or chemotherapy. any other serious pre - existing cardiovascular, pulmonary and immunological disease. wounds of the subjects included in the study underwent initial sharp debridement to remove necrotic tissue and slough as far as possible. after the debridement, foam - based dressing was done over the wounds of the study group under all aseptic conditions. an evacuation tube embedded in the foam was connected to a fluid collection canister contained within a portable vacuum / suction machine [figures 1 and 2 ]. subatmospheric (negative) pressure was applied within a range of 50 mmhg to 125 mmhg intermittently three times a day. weekly cultures were taken from the floor of the ulcers to assess for the bacterial flora. standard antibiotic regimes were administered to all the patients which consisted broad spectrum antibiotics initially and later according to the culture sensitivity report. ulcers were treated until the wound got closed surgically or spontaneously, or until completion of the 56-days (8 weeks) assessment whichever was earlier. showing application of negative pressure wound therapy showing machine used to create negative pressure complete healing was defined as 100% wound closure with re - epithelialization or scab with no wound drainage present and no dressing required. at the end of the study period patients were categorized as : complete responders : complete healing of lower limb ulcers.partial responders : 50% or greater reduction in product of the two longest perpendicular diameters from baseline.noncomplete responders : less than 50% reduction in the product of the two longest perpendicular diameters from baseline.nonresponders : no reduction in ulcer or increase in ulcer area over base line. partial responders : 50% or greater reduction in product of the two longest perpendicular diameters from baseline. noncomplete responders : less than 50% reduction in the product of the two longest perpendicular diameters from baseline. the observations were noted and all results were tabulated and analyzed by using student t - test for age, fasting blood sugar and percentage change in wound size from 1st to 8th week. the appearance of granulation tissue and the primary study end point were tested for significance by applying test. the present study was conducted in a total of 30 patients aged between 20 and 75 years of age, of either sex, having ulcer area ranging between 50 and 200 cm and fulfilling the diagnostic criteria of diabetes mellitus made by american diabetes association. the mean age of patients in group a was 61.33 7.63 years and in group b was 55.40 11.54 years. the age distribution was comparable and statistically insignificant in both the groups (p>0.10). in group a, 80% of the patients were males whereas 20% were females while in group b 86.67% of the patients were males and 13.33% were females. at first week it was observed that all the patients in group a and b had discharge from the wound. the discharge kept on decreasing over the period of observation in both the groups ; however, group a subjects had faster rate of disappearance of discharge. wound discharge was present in only 13.33% of patients in 7th and 8th week in group a as compared to 33.33% and 26.67% in group b. from the study it was observed that during the first week granulation tissue was absent in 4 patients (26.67 %) in group a and 10 patients (66.67%) in group b. it was seen that granulation tissue appeared at 2nd week in three out of four patients (75%) and 4th week in the remaining 1(25%) patient in group a (plate 4). the appearance of granulation tissue in patients of group b was at 2nd, 4th and 5th week in three (30%), 3(30%) and two (20%) patients, respectively. it was also noted that in two (20%) patients granulation tissue remained absent even at the end of observation period. this suggested early appearance of granulation tissue in patients of group a which was also found to be statistically significant the wound size showed no change in 1 (6.67%) patient of group a as compared to 3 (20%) patients of group b. it was also observed that 2(13.33%) patients of group b showed increase in wound size. the percentage decrease in the wound size was more in patients of group a as compared to group b. the mean decrease in the wound size in patients of group a was -16.14 13.04 cm and that of group b was -5.98 14.41 cm. we observed that patients of group a showed rapid clearance of bacterial load as compared to group b. this was suggested by 40% of the cultures in group a having no growth by 3rd week as compared to 20% in group b. staphylococcus aureus was the found to be most prominent in patients of group a whereas cultures from group b mostly showed mixed growth and acinetobacter. although statistically the time status of wound closure was comparable in both the groups (p>0.10), it was seen that the patients in group a showed faster healing as compared to the patients of group b. this was suggested by wounds of 9 (5 + 1 + 3) (60%) patients of group a getting closed by the end of 4th week as compared to only 3 (0 + 2 + 1) (20%) patients of group b. the patients who underwent below knee amputation were excluded from this analysis. both the groups had received similar treatment for the closure of wound, the most common mode of wound closure being stsg. although statistically the primary study endpoint was comparable in both the groups (p>0.10), group a promised better outcome (80% complete responders) as compared to group b (60% complete responders). the mean age of patients in group a was 61.33 7.63 years and in group b was 55.40 11.54 years. the age distribution was comparable and statistically insignificant in both the groups (p>0.10). in group a, 80% of the patients were males whereas 20% were females while in group b 86.67% of the patients were males and 13.33% were females. at first week it was observed that all the patients in group a and b had discharge from the wound. the discharge kept on decreasing over the period of observation in both the groups ; however, group a subjects had faster rate of disappearance of discharge. wound discharge was present in only 13.33% of patients in 7th and 8th week in group a as compared to 33.33% and 26.67% in group b. from the study it was observed that during the first week granulation tissue was absent in 4 patients (26.67 %) in group a and 10 patients (66.67%) in group b. it was seen that granulation tissue appeared at 2nd week in three out of four patients (75%) and 4th week in the remaining 1(25%) patient in group a (plate 4). the appearance of granulation tissue in patients of group b was at 2nd, 4th and 5th week in three (30%), 3(30%) and two (20%) patients, respectively. it was also noted that in two (20%) patients granulation tissue remained absent even at the end of observation period. this suggested early appearance of granulation tissue in patients of group a which was also found to be statistically significant the wound size showed no change in 1 (6.67%) patient of group a as compared to 3 (20%) patients of group b. it was also observed that 2(13.33%) patients of group b showed increase in wound size. the percentage decrease in the wound size was more in patients of group a as compared to group b. the mean decrease in the wound size in patients of group a was -16.14 13.04 cm and that of group b was -5.98 14.41 cm. we observed that patients of group a showed rapid clearance of bacterial load as compared to group b. this was suggested by 40% of the cultures in group a having no growth by 3rd week as compared to 20% in group b. staphylococcus aureus was the found to be most prominent in patients of group a whereas cultures from group b mostly showed mixed growth and acinetobacter. although statistically the time status of wound closure was comparable in both the groups (p>0.10), it was seen that the patients in group a showed faster healing as compared to the patients of group b. this was suggested by wounds of 9 (5 + 1 + 3) (60%) patients of group a getting closed by the end of 4th week as compared to only 3 (0 + 2 + 1) (20%) patients of group b. the patients who underwent below knee amputation were excluded from this analysis. both the groups had received similar treatment for the closure of wound, the most common mode of wound closure being stsg. although statistically the primary study endpoint was comparable in both the groups (p>0.10), group a promised better outcome (80% complete responders) as compared to group b (60% complete responders). the role of negative pressure dressing in healing of diabetic foot ulcers has been proposed as a novel method of manipulating the chronic wound environment in a way that it reduces bacterial burden and chronic interstitial wound fluid, increases vascularity and cytokine expression and to an extent mechanically exploiting the viscoelasticity of peri wound tissues. vac is generally well - tolerated and, with few contraindications or complications, is fast becoming a mainstay of current wound care. hence we planned to use npwt for the treatment and fast healing of diabetic foot ulcers. the demographical profile was statistically studied and found comparable with no significant difference between the groups. the mean age of patients in study group was 61.33 7.63 years and in control group was 55.40 11.54 years which was comparable to the multicenter randomized controlled trial enrolling 342 patients done by blume., who had a mean age of 58 years. the sex distribution was also similar to the above quoted study that had 79% males. we observed that there was a decreasing trend in the presence of wound discharge in both the groups. however, it was noted that the rate of disappearance of wound discharge was faster in the study group as compared to the control. only 13.33% of patients in study group had discharge at the end of 7th and 8th week as compared to 33.33% and 26.67% of patients in control group, respectively. this could be attributed to the faster rate of wound closure in the study group. in a similar study conducted by tamhankar., in four patients with mesh - related infection after abdominal wall hernia repair which were treated by npwd therapy, it was seen that npwd therapy allows salvage of infected exposed mesh by clearing the purulent discharge promoting granulation tissue formation. application of negative pressure over wound bed allows the arterioles to dilate, so increasing the effectiveness of local circulation, promoting angiogenesis, which assists in the proliferation of granulation tissue. we have also found that the patients on npwd therapy had earlier appearance of granulation tissue. of all the patients who initially did not have granulation tissue, 75% of those in the study group promised its appearance by the end of 2nd week as compared to 30% in the control group and this was also found to be statistically significant (p0.10), it was seen that the study group showed faster rate of wound closure as compared to control group. mccallon., also observed satisfactory healing in vac group in 22.8 17.4 days, compared to 42.8 32.5 days in control group. the endpoint taken was a granulated wound or a wound ready for skin grafting or healing by secondary intention spontaneously whichever was earlier. both the groups had received similar treatment for the closure of wound, the most common mode of wound closure being stsg [figures 3 and 4 ]. it was also observed that the failure rate was higher in patients of control group as compared to study group., who had observed that npwt delivered by vac device was safe and effective treatment for complex diabetic foot wounds and could lead to higher proportion of healed wounds, faster healing rates and potentially fewer re - amputations than standard care., have also reported overall progressively increasing wound debridementdepth and amputation rates in control groups ; however the same increasing trend didnot occur in the npwt group. diabetic ulcer treated with negative pressure wound therapy postoperative picture of the healed ulcer at the end of the study, although the primary endpoint was statistically comparable in both the groups (p>0.10), the study group promised a better outcome (80% complete responders) as compared to the control group (60% complete responders) [table 1 ]. analyzing the results of our study, we opine that npwt has a definitive role in promotion of proliferation of granulation tissue, reduction in the wound size, rapid clearing of the wound discharge and bacterial load. our data demonstrates that negative pressure wound dressings decrease the wound size more effectively than saline gauze dressings over the first 4 weeks of therapy. it is suggested that npwt is a cost - effective, easy to use and patient - friendly method of treating diabetic foot ulcers which helps in early closure of wounds, preventing complications and hence promising a better outcome. | introduction : foot disorders such as ulceration, infection and gangrene are the most common, complex and costly sequelae of diabetes mellitus.[13 ] even for the most superficial wounds, treatment is often difficult with poor healing responses and high rates of complications. the purpose of this study is to compare the rate of ulcer healing with the negative pressure dressing technique to conventional moist dressings in the treatment of diabetic foot ulcers.materials and methods : the study was conducted on 30 patients, which were divided into two groups. one group received negative pressure dressing while other group received conventional saline moistened gauze dressing. results were compared for rate of wound healing.results:there was a statistically significant difference in the rate of appearance of granulation tissue between the two groups ; with granulation tissue appearing earlier in the study group. the study group promised a better outcome (80% complete responders) as compared to the control group (60% complete responders).conclusions : negative pressure wound therapy has a definitive role in healing of diabetic foot ulcers. |
ocular itching associated with allergic conjunctivitis is a common patient complaint, and up to 40% of the us population is affected by allergic conjunctivitis, according to epidemiologic surveys.1 although there are masqueraders of allergic conjunctivitis, including bacterial etiologies that produce the common papillary conjunctival reaction, diagnosis is generally straightforward. typical exam findings, including conjunctival injection and papillae, combined with standard complaints of seasonal- or exposure - related ocular itching, lead to the clinical diagnosis. the disease begins with antigen exposure, which stimulates mast - cell degranulation, histamine release, and stimulation of a downstream inflammatory cascade.2 management of the condition depends on several considerations. first, an oral agent may be used when allergic rhinitis or another systemic symptom is also present. even in this case, however, eye drops may be necessary to control the ocular itching, despite systemic therapy. second, there are a number of ophthalmic drugs available, with different mechanisms of action, dosing frequency, and tolerability. finally, there is a wide range in price points for the various eye drops, including on - patent and generic formulations as well as prescription and over - the - counter alternatives. available oral h1-antihistamines include fexofenadine (allegra and allegra - d, prescription [rx ] only), loratidine (claritin and others, over - the - counter [otc ]), desloratidine (clarinex, rx only), and cetirizine (zyrtec, otc).3 ophthalmic preparations, which tend to have a more rapid onset than oral agents with respect to ocular itching,2 include a number of drugs with antihistamine activity (h1-antagonists) and varying degrees of mast - cell stabilizing activity. these preparations include azelastine (optivar, rx only), epinastine (elestat, rx only), ketotifen (alaway, zaditor, and others, otc), and olopatadine (patanol, pataday, rx only) (see table 1).4 older agents that act primarily as mast - cell stabilizers are also available, including cromolyn (crolom, opticrom, rx only), nedocromil (alocril, rx only), lodoxamide (alomide, rx only), and pemirolast (alamast, rx only).4 bepotastine besilate 1.5% (bepreve, ista pharmaceuticals, rx only) is a recently us food and drug administration (fda)-approved ophthalmic h1-antihistamine, and the available literature concerning its use will be reviewed. in addition to antihistamines and mast - cell stabilizing drugs, various ophthalmic corticosteroids are available for the treatment of allergic conjunctivitis. their use is typically limited to severe disease due to their more impressive side effect profile, which generally includes cataract formation and elevation of intraocular pressure. loteprednol etabonate 0.2% suspension is a topical ester corticosteroid2 that has been found to exhibit a low risk of intraocular pressure elevation over 4 weeks of therapy5 while reducing the symptoms of seasonal allergic conjunctivitis over a similar period of follow - up.68 due to the combination of effectiveness and a low side effect profile, loteprednol may become a useful alternative to antihistamine therapy even in mild to moderate allergic conjunctivitis, especially when prescribed as a short - term course. other, more potent corticosteroids, such as prednisolone acetate 1% solution, will likely remain in use for more severe disease. bepotastine besilate 1.5% ophthalmic solution (bepreve, rx only) is an h1-antihistamine that was recently granted fda approval as a new treatment for itching associated with allergic conjunctivitis. tanabe seiyaku co., ltd. and prepared as an oral medication for allergic rhinitis, receiving approval in japan in july 2000.9 development of the ophthalmic solution for us release was by ista pharmaceuticals, and approval was granted in 2009 based on data from japanese development programs as well as two domestic conjunctival allergen challenge studies, including a single - site phase ii / iii trial and a multisite phase iii study. available data concerning the pharmacology, clinical use, and safety of this drug will be reviewed. bepreve, produced by ista pharmaceuticals, is a clear, colorless to pale yellow solution containing bepotastine besilate as the active ingredient in a 1.5% solution, with a ph of 6.8 and osmolality of 290 mosm / kg.9 this solution also contains benzalkonium chloride 0.005% as a preservative.9 bepotastine is an h1-antihistamine and an inhibitor of histamine release from mast cells.10 it is a piperidine derivative, similar to fexofenadine, ebastine, and loratidine.11 multiple anti - inflammatory effects have been demonstrated, possibly as downstream mediators of the antihistamine activity. for example, in vitro studies suggest that bepotastine specifically suppresses proinflammatory cytokine production by keratinocytes, including inhibition of cd54 expression.12 recent work on guinea pigs showed that bepotastine, along with several other h1-antihistamines, reduces vascular hyper - permeability in both antigen - induced and histamine - induced hyperpermeability models.13 this work also showed that bepotastine inhibits in vitro eosinophil chemotaxis induced by leukotriene b4, and pretreatment with bepotastine limits conjunctival eosinophil infiltration after topical platelet - activating factor instillation.13 the pharmacokinetic properties of bepotastine as an ophthalmic solution are described in phase i trial data from japan, and these will be described as reported in the fda office of clinical pharmacology review of the japanese data.9 first, a repeated instillation study was performed, testing four - times - a day dosing in both eyes for 7 days in 12 healthy adult male subjects, half of whom instilled bepotastine besilate 1.0% and half instilled the 1.5% formulation. venous blood samples were measured by high - performance liquid chromatography and demonstrated a bepotastine plasma concentration peak 12 hours postinstillation. the mean maximum concentration (cmax) for the 1.5% group was 7.3 1.9 ng / ml, which was much lower than the cmax seen in the phase i single oral dose trial, even at the lowest tested oral dose (cmax was 22.4 2.1 ng / ml for the 2.5 mg oral dose). at the clinically relevant, approved japanese oral dose of 10 mg, the cmax was 101.3 3.5 ng / ml, which is over 13 times higher than the cmax seen in the repeated ophthalmic dosing trial. thus, although there is systemic absorption of the ophthalmic drop, the plasma concentrations are quite low, minimizing the likelihood of systemic adverse effects. furthermore, plasma concentrations at 24 hours postinstallation were below the quantifiable limit of 2 ng / ml in 11 of 12 subjects. in the oral single - dose study, 75%90% of the administered dose was secreted in the urine as unchanged drug by 24 hours after administration within the 2.540 mg dose range. an additional phase i study addressed the metabolism of bepotastine by liver microsomes, showing that there was minimal metabolism by cyp3a4, cyp2c9, and cyp2c19, again as reported in english by the fda office of clinical pharmacology review of the japanese data.9 within the relevant concentration range, it was concluded that bepotastine would likely have no effects on concomitantly metabolized drugs involving these enzymes. finally, a protein - binding phase i study was performed, demonstrating 55.4% mean plasma protein binding of the drug 12 hours after a 10 mg oral dose.9 this binding level was independent of plasma drug concentration. there have been two randomized, double - masked, placebo - controlled trials of bepotastine besilate ophthalmic solution as a treatment for ocular itching associated with allergic conjunctivitis.14,15 both studies evaluated the effectiveness of the drug in comparison with its vehicle in a conjunctival allergen challenge (cac) model. no comparative human clinical studies against other drugs have been performed, although bepotastine performed well compared with other h1-antihistamines in an experimental model of allergic conjunctivitis in guinea pigs.13 the cac model employed was consistent between the two studies.14,15 subjects were included on the basis of age (10 years) and a positive skin - test reaction to an allergen within the prior 2 years. further, during two screening visits, subjects had to demonstrate a positive reaction to the testing protocol with respect to ocular itching and conjunctival redness. visit 1 (day-21) was used to determine the lowest dose of the defined antigen (from skin testing for that patient) that would elicit an ocular allergic response. the primary outcome variables measured were subject - evaluated ocular itching at 3, 5, and 7 minutes post - cac (04 scale allowing half steps) and investigator - evaluated conjunctival redness at 7, 15, and 20 minutes post - cac (04 scale allowing only whole steps). patients with validated responses to the cac continued with the study protocol. at visit 3 a (day 0), patients were randomly assigned to bepotastine besilate 1.0% ophthalmic solution, bepotastine 1.5%, or inactive vehicle (placebo), and one drop of the appropriate solution was instilled in each eye. this time period was chosen to correspond to a duration of action suitable for once - daily dosing. primary as well as secondary outcome measures were obtained at stated intervals, with grading of allergic conjunctivitis over the next 20 minutes. visit 4 (day 14) was scheduled to give a 2-week washout of the study drug instilled on day 0. at this visit, the appropriate drug was again instilled, and a cac was administered after 8 hours and its effects graded. this time period was chosen to correspond with a suitable duration of action for a drug intended to be dosed twice daily. visit 5 (day 28) was identical to visit 4, except cac was administered at 15 minutes after the drug instillation. the first study was a phase ii / iii single - center cac study that analyzed 107 subjects evenly divided among the three study groups.14 analysis of the primary outcomes, ocular itching, and conjunctival redness was applied to the per - protocol (pp) population that excluded protocol violators and to the intention - to - treat (itt) population using last observation carried forward (locf) for missing data. clinical significance was defined in the protocol as 1.0-unit between - group (placebo versus drug) difference between mean ocular itching or conjunctival redness at two of three time points at a study visit, and 0.5-unit mean difference at all time points. statistical analysis of the conjunctival hyperemia data showed statistically significant improvements in hyperemia scores for both concentrations of drug compared with placebo (0.20.8 u difference, depending on time point, for bepotastine 1% versus placebo ; 0.10.6 u difference for bepotastine 1.5%), but these improvements did not meet the predefined criteria for clinical significance. ocular itching data, however, showed statistically and clinically significant improvements with both concentrations of bepotastine for the 15-minute (rapid onset of action) and 8-hour (twice - a - day dosing) cac. bepotastine 1.5% showed a similar effect at both of these time points (1.21.5 u for the 15-minute cac, 1.21.7 u for the 8-hour cac), but bepotastine 1.0% showed a drop - off in the effect with the longer duration (1.31.4 u for the 15-minute cac, 0.91.1 u for the 8-hour test). bepotastine 1.5% also demonstrated a significant effect in clinical significance for the 16-hour cac (0.91.0 u). the second efficacy study was a phase iii multicenter, prospective, double - masked, placebo - controlled, cac clinical trial.15 this study enrolled 130 subjects at five clinical sites, dividing the subjects equitably between the three groups. study methodology was identical to the single - center trial, except ocular comfort was graded by the subject on a 4-point scale immediately after drop instillation and 5 minutes later. in this study, both concentrations of bepotastine demonstrated at least a 1.2-unit reduction in ocular itching for the 15-minute and 8-hour cacs. efficacy was significantly reduced for both concentrations at the 16-hour time point. a modest reduction in conjunctival redness was also seen, but only during the 15-minute cac. there appears to have been a small decrease in ocular comfort for each drug concentration compared with placebo, although the measure approaches zero for all subjects at all time points, indicating relatively good ocular comfort on drug administration. the fda statistical analysis evaluated the robustness of the submitted efficacy data.16 the danger of using locf for missing data was explored, noting the potential biasing of results with this method. sensitivity analyses using alternative population sets (pp and itt with observed data only) and repetition of the itt analysis with different imputation methods for missing data showed that bepotastine 1.0% and 1.5% maintained a predefined standard of clinical significance for ocular itching, satisfying this critique. in addition, the choice of p values for clinical significance evaluation was criticized for the lack of a multiplicity adjustment to account for the majority of time points stipulation that allows multiple ways to satisfy the clinical significance criteria. when these adjustments were made, however, the efficacy conclusions remained unchanged. in summary, bepotastine 1.0% and 1.5% are found to be efficacious for the treatment of ocular itching related to allergic conjunctivitis, with a swift onset of action and appropriate duration of action for twice - a - day dosing. this drug does not appear to be sufficiently effective for the indication of conjunctival redness. finally, bepotastine 1.5% solution was found to be more effective than the 1.0% concentration, so only the 1.5% concentration has been fda approved. the side effect profile of bepotastine ophthalmic solution is similar to that of other ocular antihistamines, a list of uncommon or low - risk events comprising headache, asthenia, blurry vision, burning on drug instillation, cold and flu symptoms, cough, fatigue, dry eye, foreign body sensation, eyelid edema, keratitis, hyperemia, nausea, pharyngitis, pruritis, rhinitis, sinusitis, sore throat, and bitter taste.17 tolerability was reported for each of the randomized trials.14,15 in addition, an unpublished safety study was undertaken to evaluate only the 1.5% bepotastine concentration at six sites in the us, and this was completed with a randomized, double - masked, placebo - controlled, parallel - group safety study design. results of this study will be given as reported in fda documents.16,17 in the single - site phase ii / iii trial, the number of ocular adverse events reported was greater in the placebo group (8.3% of subjects) than in either treatment arm of the study, and all events were limited to eye irritation, foreign body sensation, and a single conjunctival cyst in the bepotastine 1.5% group.14 although a small number of nonocular adverse events were reported in all study groups, including placebo, only taste on instillation appears to be more common in the drug groups than in the placebo arm (20%25% incidence in the two drug concentration groups versus 0% for placebo).14 ocular and nonocular side effects were similar in the multicenter efficacy trial, revealing no new concerns, and in this instance a taste on instillation was only noted in 5% of subjects in the drug arms versus 0% in the placebo arm.15 the dedicated safety study for bepotastine 1.5% tested bilateral twice - a - day dosing for 6 weeks in healthy volunteers 3 years old.16,17 six sites enrolled 861 healthy subjects, randomizing to a 2:1 active : vehicle ratio. the population was predominantly caucasian (85% of 575 subjects in the bepotastine group and 84% of 286 subjects in the vehicle group), and females outnumbered males the pediatric population included 47 bepotastine and 25 vehicle subjects in the 39 year age group, as well as 40 bepotastine and 15 vehicle subjects in the 1017 year age group. the study consisted of four visits over approximately 43 days, and subjects completed a diary to document drug instillation. ninety - three percent of subjects (801 of 861) completed the entire study protocol. this study demonstrated the safety of bepotastine 1.5% ophthalmic solution, with no deaths and no serious adverse events reported. there were 12 subjects who withdrew from the study due to an adverse event, six from the bepotastine group and six from the vehicle group. the adverse events that appeared to be potentially related to the study drug were eye irritation (4.7% bepotastine versus 2.1% vehicle), taste perversion (14.6% versus 1.4%), bad taste (7.8% versus 0.3%), headache (3.5% versus 2.4%), and a variety of rarely reported nasal complaints (eg, nasal congestion, postnasal drip, sneezing). the most commonly reported adverse effect was a taste - related event in 25.2% of subjects, a result that was highly statistically significant.16 subjects taking bepotastine did not demonstrate any clinically significant change from baseline or compared with the vehicle in any other safety measurements, including endothelial cell counts, intraocular pressure, visual acuity, slit - lamp biomicroscopy, and dilated fundoscopy. in the 39 year age group subject population, only four adverse events were reported, all related to taste.17 the results for the 1017 year age group were similarly encouraging, with six adverse events related to taste, one to headache, and one minor ocular complaint (compared with two similar ocular complaints in the vehicle group).17 finally, there were no statistically significant differences in ocular comfort between the study drug and its vehicle in this study. it is noted that bepotastine has been available as an oral medication since 2000 in japan. as reported in the fda clinical review,17 japanese postmarketing experience demonstrates an adverse event rate similar to that found during clinical studies prior to drug approval (9.47%), with the two most common events being drowsiness (1.32% of patients) and upper abdominal pain (0.13%). a retrospective review of bepotastine oral tablets was also conducted in the pediatric population (ages 514 years), and all confirmed events were found to be of the mild variety, including drowsiness (0.4%), thirst (0.2%), and urticaria (0.2%). a comparison study in healthy adults showed that bepotastine had a low rate of sedation even among a group of later - generation h1-antihistamines.18 according to a large patient survey, the sedative effects of oral bepotastine appear to be in line with other second - generation h1-antihistamines when evaluated using a visual analog scale.19 in summary, over a 6-week time period, twice - daily bepotastine besilate 1.5% ophthalmic solution was found to be a safe drug for subjects 3 years old when dosed bilaterally. no serious adverse events were observed, although abnormal taste complaints were common, found in approximately 25% of subjects across studies. ocular comfort does not appear to be compromised by bepotastine. although drowsiness is a common complaint due to systemic h1-antihistamines, this was not seen after ophthalmic dosing. finally, bepotastine ophthalmic solution has not been tested in children under 3 years old or in pregnant women. the recent fda approval and commercial availability of bepotastine besilate 1.5% ophthalmic solution adds to the array of treatments available for ocular itching associated with allergic conjunctivitis. although this drug has not been directly compared with other available treatments, studies to date prove its effectiveness as well as its mild side effect profile. the twice - daily dosing strategy, although less optimal than the once - daily formulation of olopatadine 0.2% (pataday), should lend itself to reasonable adherence and is identical to the dosing of azelastine, epinastine, ketotifen, and the 0.1% solution of olopatadine. a key disadvantage to bepotastine is that its formulation, like most other drugs for this condition, contains benzalkonium chloride, which can be absorbed by contact lenses and is an ocular irritant to which certain patients may be allergic. there are no preservative - free formulations of ophthalmic h1-antihistamines, so this drawback does not distinguish bepotastine. the expense of the various ophthalmic h1-antihistamines is displayed in table 2, with a column showing the price per day of bilateral treatment with each agent. calculating based on retail costs, the over - the - counter ketotifen formulations are clearly the least expensive options within this class, with daily expenses of $ 0.26 (alaway) or $ 0.56 (zaditor). for patients who do not respond to ketotifen or who are unable to tolerate the formulations of these agents, generic azelastine bepotastine is the next least expensive ophthalmic h1-antihistamine, at $ 3.20 per day of therapy. the least expensive on - patent h1-antihistamine formulation is bepotastine 1.5%, at $ 3.55 per day. this compares quite favorably with other on - patent prescription agents, such as olopatadine 0.1% ($ 4.66/day) and olopatadine 0.2% ($ 4.49/day). bottle size may be another advantage of bepotastine, which is marketed as a 10 ml volume. as the days / bottle column in table 2 displays, this provides approximately 50.1 days of therapy in a single bottle, which is equal to the largest size of ketotifen and longer than any other agent. this length of time may encompass the entire season of allergic conjunctivitis for some patients who display symptoms in only part of the year. these patients would only need to purchase a single bottle of bepotastine each year, which is a distinct efficiency advantage over smaller bottle sizes. a review of the available literature regarding bepotastine besilate 1.5% ophthalmic solution (bepreve) confirms that it is a safe and effective agent for the treatment of ocular itching associated with allergic conjunctivitis. this agent does not eliminate conjunctival redness, and it should not be used in children under 3 years old or in pregnant women. aside from these concerns, bepotastine could be widely prescribed and should prove to be an effective alternative to available therapies, although there are no comparative clinical data to demonstrate any difference in effectiveness between bepotastine and other ophthalmic h1-antihistamines. although the price point for bepotastine is higher than the over - the - counter ketotifen formulations, the current retail price is lower on a per - day - of - therapy basis than the retail price of any other on - patent drugs within bepotastine s class. providers should feel confident in prescribing bepotastine for ocular itching associated with allergic conjunctivitis, especially for patients who have failed less expensive alternatives. | bepotastine besilate 1.5% solution is an h1-antihistamine recently approved by the food and drug administration for the topical treatment of ocular itching associated with allergic conjunctivitis. several clinical studies have demonstrated its safety as well as its efficacy versus placebo. this review finds that bepotastine besilate 1.5% solution is a suitable alternative to other agents within the class of h1-antihistamines, but there are no clinical trial data to suggest that it holds any specific advantages over other agents. |
there is substantial evidence that adaptation to different environments can lead to the evolution of reproductive isolation between populations, a process referred to as ecological speciation [16 ]. prezygotic isolation can evolve as mating signals and preferences adapt to different environments [712 ]. extrinsic (environmentally dependent) postzygotic isolation may also evolve since hybrids have intermediate phenotypes and are poorly adapted to parental habitats [1317 ]. currently, there is less evidence that genetic incompatibilities between populations (intrinsic postzygotic isolation) can evolve simply as a consequence of adaptation to different habitats [1820 ]. most identified intrinsic isolating barriers have no clear relationship to adaptation and may have arisen subsequent to ecological divergence [2123 ]. however, theoretical and empirical work suggests intrinsic isolation can arise through ecological divergence if there are epistatic interactions between alleles conferring environment - specific adaptations [2426 ]. when prezygotic, extrinsic, and intrinsic postzygotic reproductive isolating barriers evolve as byproducts of adaptation, the probability that they will lead to speciation depends on their cumulative strength and ability to persist in the face of gene flow when incipient species come into contact. if the cumulative strength of isolating barriers is insufficient, population divergence will be lost via introgression, and speciation will not occur. therefore, determining how adaptation generates both pre- and postzygotic isolating barriers and how rapidly these barriers evolve is a key focus of speciation research. much previous work has focused on timing and order in which reproductive isolating barriers arise, but has given little consideration to their ecological context. in species of drosophila studied in a common laboratory environment, prezygotic isolation evolves faster than postzygotic isolation [29, 30 ]. however, this effect seems to be driven by the effect of sympatry and, in allopatric species, pre- and postzygotic isolation evolve at the same rate. prezygotic isolation also evolves well before postzygotic isolation in birds [31, 32 ], salamanders, and several groups of fish (including centrarchids, african rift lake cichlids, and darters [35, 36 ]). for instance, postzygotic isolation in fish appears to accumulate slowly with hybrid inviability not becoming complete until species have been separated for 10 to 20 million years [33, 34 ]. however, in many of these studies, hybrids are raised in a common laboratory environment, which may underestimate hybrid inviability. differences in population ecology and how these may relate to the strength of isolating barriers are not usually considered. one exception to this is work on stickleback fish which has found that young stickleback species pairs exhibit prezygotic and environmentally based postzygotic isolation, while older pairs show both prezygotic and intrinsic postzygotic isolation [8, 13, 37, 38 ]. in our study, we ask which reproductive isolating barriers have evolved between ecologically divergent populations within one species of killifish and compare them to barriers that have evolved between two sister species. this allows us to determine the order in which isolating barriers arise as populations adapt to different ecological conditions. the rainwater killifish, lucania parva, is a euryhaline species with permanent populations existing in fresh, brackish, and salt water across the southeastern united states. l. parva 's sister species, the bluefin killifish (lucania goodei), is found almost exclusively in fresh water in florida. sympatric populations of l. parva and l. goodei can be found in several freshwater sites across florida. multiple lines of evidence suggest that adaptation to different salinity conditions has occurred between species. l. goodei has higher fitness in fresh water relative to l. parva, and l. parva fares better in brackish and salt water than l. goodei. additionally, l. goodei has a decreased rate of hatching success at high salinities while l. parva has a lower rate of survival to adulthood in fresh water [4143 ]. however, l. parva appears to have equal hatching success with l. goodei in fresh water. all this previous work on l. parva and l. goodei has raised eggs with the fungicide methylene blue. while this fungicide improves hatching success, it may do so disproportionally for different salinities, populations, or species. therefore, in our study, we raised eggs in water with and without methylene blue. behavioral isolation is quite strong with l. parva and l. goodei mating pairs taking longer to produce eggs than conspecific pairs and producing fewer eggs. backcrosses, f1, and f2 hybrids have reduced survival, particularly at high salinities. in addition, f1 hybrids sons of l. parva females and l. goodei males have reduced fertility. some of these isolating barriers between l. parva and l. goodei may have arisen due simply to adaptation to fresh and salt water. life in fresh water and salt water pose different osmoregulatory challenges for aquatic animals. in fresh water, fish need to keep excess water out of their bodies, while retaining vital salts. however, marine fish need to extricate salt, but retain water. throughout the life of the fish, osmoregulation can occur in the gills, guts, kidneys, and skin. therefore, adaptation to salinity can potentially cause divergence in many genes involved in ion regulation [47, 48 ], increasing the likelihood of speciation as a direct consequence of adaptation to salinity. to ask how salinity may drive the evolution of isolation barriers in lucania, we measured isolation between l. parva populations adapted to different salinity environments. we collected l. parva from a permanent fresh water population (pecos river) and a salt water population (indian river lagoon). we crossed pecos and indian river fish and predicted that if prezygotic isolation existed, between populations, mating pairs would take longer to mate and produce fewer eggs than within population pairs. we then raised pecos - indian river hybrid eggs in five water chemistries ranging from fresh to salt water and measured survival. if postzygotic isolation exists, hybrid eggs and fry should have lower survival than either of the parental populations. hybrid inviability across environments would be evidence for intrinsic isolation, while environmentally dependent inviability would suggest that isolation is extrinsic. furthermore, if any local adaptation is present, we would predict the freshwater population to have higher survival than the saltwater population in fresh water treatments and the saltwater population to have higher survival in salt water conditions. we measured survival of our l. parva populations with and without methylene blue to determine if the fungicide had any effect on measures of postzygotic isolation. additionally, we wished to compare the survival of freshwater, saltwater, and hybrid l. parva to l. goodei survival. previous work has established that l. goodei has extremely low survival in salt water, but equal survival with l. parva in fresh water. however, in these studies, eggs were raised in methylene blue, and only a single fresh water treatment was used. therefore, we collected eggs from one population of l. goodei and raised them in two fresh water treatments in the absence of methylene blue. we predicted that l. goodei should have higher survival in fresh water than l. parva. we collected l. parva from two ecologically different and geographically distant sites : an inland river in texas and the atlantic ocean off the coast of florida. our freshwater site was pecos river, along the pecos - crockett county border, tx. at the time of collection, the carbonate hardness (kh, a measure of mineral content) of the water was low (between 3 and 4). however, the upper pecos river does have a history of salinization due to input from salt springs and dam construction altering water flow, which may contribute to l. parva 's persistence there. our saltwater site was indian river lagoon, brevard county, fl, on the atlantic coast. the collected fish were transported back to university of illinois and housed in 75109 l stock tanks. indian river (ir) fish were kept in reverse osmosis water raised to 35 ppt salinity using instant ocean sea salt (spectrum brands, atlanta, ga). they were then transitioned to 10 ppt water, then at the beginning of the experiment to tanks containing city water treated with the dechlorinating agent start right (jungle laboratories, cibolo, tx) at 2 ppt salinity. pecos river fish were kept in treated city water at 2 ppt salinity and with alkaline regulator (seachem, madison, ga) added to bring the carbonate hardness (kh) to 10. fish were fed daily ad libitum with a mixture of frozen brine shrimp and flake food. fish were maintained under a light cycle of 14 hours light, 10 hours dark. we set up four different cross types : two within population crosses (pecos female by pecos male, ir female by ir male) and two between population crosses (pecos female by ir male, ir female by pecos male). there were 8 replicates of each cross type, for a total of 32 pairings. for each pair, we placed one male and one female in a 38 l tank filled with dechlorinated city water at 2 ppt salinity. four yarn mops were provided as a spawning substrate (two floating and two sinking mops). all collected eggs were checked under a microscope to verify that they were recently fertilized. killifish eggs take approximately 79 days to hatch ; therefore, most eggs (at 1 - 2 days old) were very early in development when they were transferred to their water treatments. latency to mate was measured over the first 47 days and was calculated as the number of days until the first egg was found. if a pair had not mated after 47 days, we assigned them a latency of 48 days (the total number of days plus 1 day). after 47 days, we removed visual barriers between tanks to encourage spawning and continued collecting eggs. we summed the total number of eggs laid over the entire experiment (61 days). there were three fresh water treatments : pure reverse osmosis water (ro), soft water (kh3), and hard water (kh8). the ro water was created using a filtration system that removes sediment, chlorine, and other large ions from city water (aquafx barracuda 4 stage ro / di system, winter park, fl). soft water was created by adding alkaline regulator (seachem, madison, ga) and r / o right (kent marine, franklin, wi) to adjust the ionic content of ro water to a carbonate hardness of kh3. hard water was created by adding alkaline regulator and r / o right to dechlorinated city water until its hardness was kh8. the salt water treatments were made by adding instant ocean sea salt to ro water until the desired salinity was reached. ocean water is typically 32 ppt, and we used two salinity treatments : saline (20 ppt) and hypersaline (40 ppt). additionally, we raised some eggs in the kh8 and 20 ppt treatments with methylene blue, the antifungal agent. a 3 ppm solution of methylene blue (c16h18n3scl ; kordon llc, hayward, ca) was added to the water immediately after eggs were placed in it. we rotated the water treatment every egg collection day to assure an equal distribution of eggs in each water treatment. we collected eggs until each water treatment had at least 10 eggs from each tank. once the eggs hatched, we transferred the fry into clean tubs with the same water treatments. we recorded the number of eggs that were alive or dead, the number of eggs hatched, and the number of fry that were alive or dead. these censuses continued until 14 days after hatching, at which point fry were euthanized with an overdose of ms-222. we measured the proportion of eggs that hatched (hatching success), the proportion of fry that survived to 14 days of age (fry survival), and the proportion of eggs that produced surviving fry of 14 days of age (total survival). we combined the data for the two between population cross types into one hybrid group. we had eggs from 32 families total (pecos = 8, ir = 8, hybrid = 16). not all families had eggs in all water chemistries ; therefore, we list sample sizes for each water chemistry in our table legends. to measure l. goodei survival in fresh water in the absence of methylene blue, we collected eggs from l. goodei stock tanks (not from the preestablished crosses). the eggs collected were placed into kh3 or kh8 treatments, both without methylene blue. these eggs were also checked under a microscope to verify they were fertilized. hatching success and fry survival all statistical analyses were performed using sas statistical software (sas v 9.1, cary, nc). measures of prezygotic isolation (latency to mate and total number of eggs produced) were analyzed in a general linear model with male source population (pecos, indian river), female source population (pecos, indian river), and the interaction between male population and female population. if behavioral isolation existed, we would expect a significant interaction between male and female population. there was an outlier in our latency to mate data, with one indian river female by pecos male taking more than 47 days to mate, so we performed the analysis with and without this outlier to determine if it affected our conclusions. for survival data, we analyzed the proportion surviving at each life stage for each cross type using generalized linear models assuming a binomial distribution (proc genmod in sas) and used maximum likelihood to evaluate the significance of effects. to determine survival in the absence of methylene blue, we used a model that considered the effects of water chemistry (ro, kh3, kh8, 20 ppt, 40 ppt), cross type (pecos, ir, hybrid), and their interaction on the probability of hatching, fry survival, and total survival. we also ran analyses where we included family (nested within cross type) as a repeated factor in our general linear model, but it did not alter our results, and these analyses are not presented here. to determine the effects of methylene blue on survival in hard and 20 ppt, we ran a second model which examined the effects of cross type, water chemistry (kh8, 20 ppt), presence / absence of methylene blue and their interactions on the probability of hatching, fry survival, and total survival. to compare l. parva to l. goodei fresh water survival in the absence of methylene blue, we analyzed probability of survival at each stage (egg, fry, total) in fresh water chemistries (kh3, kh8), based on cross type (pecos, ir, hybrid, l. goodei) and included the interaction between cross type and water chemistry. means and standard errors we found no evidence for prezygotic isolation between pecos and indian river fish. between populations pairs (pecos male by indian river female ; indian river male by pecos female) did not differ from within population pairs in latency to mate or total number of eggs produced (table 1 ; figure 1). however, when the outlier was removed, we found there was a difference in latency to mate between female populations with indian river females mating sooner than pecos females (ir = 5.00 + 1.89 days, pecos = 9.94 + 5.82 days). despite a lack of prezygotic isolation, we found that offspring from pecos - indian river hybrid crosses had reduced survival. hybrid eggs had lower hatching success than within population eggs across different water treatments (table 2(a), figure 2(a)). the proportion of fry that lived and total survival were also lower for hybrid crosses, but only in hard water (tables 2(b) and 2(c) ; figures 2(b) and 2(c) ; significant cross by water treatment interaction). however, no reduction in hybrid hatching rates was detected when methylene blue was added to the water treatments (table 3, figure 3 ; significant methylene blue by cross interaction). there was little evidence for local adaptation in egg and fry survival as we did not detect consistent differences between pecos and indian river survival. in both populations, l. goodei eggs hatched more than l. parva eggs in fresh water treatments in the absence of methylene blue (table 4 ; figure 4). total survival of l. goodei eggs and fry was also higher than l. parva survival in fresh water. these differences seem primarily driven by high survival of l. goodei eggs and fry in soft water treatments. these results are in contrast to previous work that found no difference between the species when methylene blue was used. here, we show that postzygotic isolation has begun to evolve between freshwater and saltwater populations of l. parva this suggests that genes involved in hatching success and fry survival evolve more rapidly between l. parva populations than genes involved in mating traits and preferences. most previous work suggests that pre- and postzygotic isolation evolve at similar rates in allopatric populations [29, 30 ], but this does not appear to be true in l. parva. when we examined population differences within l. parva, we found that f1 hybrids between freshwater and saltwater populations had reduced survival compared to offspring from within population crosses. these effects were most apparent in challenging water chemistries : in fresh water and in the absence of methylene blue. the lethality of hard water may be due to fungus that grew readily in this water treatment. fungal infections are a major source of egg mortality and both high salinity and methylene blue can prevent infection, although methylene blue is more effective [44, 50 ]. methylene blue may also add ions to the water, which may decrease osmoregulatory stress and may be why methylene blue also increased fry survival at low salinities. this suggests that hybrid eggs were less viable than eggs from within population crosses and physiologically challenging water chemistries revealed this decreased viability. we also showed that l. parva has lower survival compared to l. goodei in fresh water in the absence of methylene blue. this contradicts previously published results that used methylene blue and found no difference in fresh water survival between species [4143 ]. this suggests that f1 hybrids between l. goodei and l. parva may also have low survival in fresh water, but these effects have been masked by the use of methylene blue in previous studies and hybrid fitness may have been previously overestimated. the decreased viability of pecos and indian river hybrid offspring suggests that intrinsic postzygotic isolation exists between populations. the main difference between pecos and indian river populations is their native salinity, suggesting that genetic incompatibilities have arisen as a byproduct of adaptation to saline environments. however, in allopatric populations, any mechanism which causes unique alleles to become fixed has the potential to cause incompatibilities as novel alleles come into contact and interact in hybrids (dobzhansky - muller incompatibilities : [18, 5153 ]). intrinsic postzygotic isolation between populations can also arise due to genetic drift [20, 52, 54 ] or genomic conflict. pecos and indian river are geographically distant, separated by more than 2400 km and the gulf of mexico. work on other related species from the fundulus clade has found substantial divergence between east and west gulf populations, possibly due to genetic drift. therefore, we are currently working on determining the degree of genetic divergence and phylogenetic relationship between these populations using sequence data. in addition, we are conducting crosses between other ecologically divergent populations from the same geographical region as well as geographically distant but ecologically similar populations. this ongoing work will determine if hybrid inviablity in l. parva evolves primarily due to salinity adaptation rather than due to drift. how isolation arises during the initial stages of speciation, when a single population splits into two and populations begin to diverge, still represents a missing link in speciation research. by showing that intrinsic postzygotic isolation has begun to evolve between divergent l. parva populations, our work suggests it may have been the first barrier to arise between l. parva and its sister species l. goodei. adaption to salinity is primarily physiological and, therefore, may be particularly likely to cause intrinsic isolation through epistatic interactions. similarly, physiological changes associated with toxic environments also appear to lead to substantial genetic changes between populations and, in some cases, to hybrid inviability [5861 ]. therefore, physiological adaptation may be a primary force leading to postzygotic incompatibilities. currently, there are competing ideas about how isolating barriers evolve during speciation. in one proposed scenario, thus, prezygotic isolation plays a primary role in preventing interbreeding, and postzygotic isolation slowly completes the process of speciation as decreased hybrid fitness and irreversible genetic incompatibilities accumulate [28, 62, 63 ]. however, this conclusion is based on studies of species pairs that have already undergone speciation [29, 34, 35 ], populations that occur in sympatry where reinforcement may have strengthened prezygotic isolation [30, 64, 65 ], or populations in which feeding and mating occur in the same habitat (such as phytophagous insects [6668 ]). nevertheless, some incipient species do show prezygotic isolation without any postzygotic barriers [57, 69, 70 ].. genetic divergence might produce hybrid inviability between populations and prezygotic isolation evolves subsequently as divergence continues or as incipient species come into sympatry and reinforcement occurs. when natural selection drives genetic divergence between populations, evolving postzygotic isolation should be primarily environmentally dependent. many examples of adaptation to divergent environments producing extrinsic isolation exist [1, 4, 15, 17, 19, 7173 ], while there are few examples for intrinsic isolation. in a survey of 20 ecologically divergent species pairs, all species exhibited some prezygotic isolation and extrinsic postzygotic isolation, but only three pairs had any documented intrinsic postzygotic isolation. intrinsic isolation as a result of ecological divergence has only been substantially documented in dwarf and normal lake whitefish [74, 75 ], copper tolerant plants, and an experimental evolution study in yeast. however, few studies distinguish between extrinsic inviability and intrinsic inviability that appears under stressful conditions, such as the decreased viability that appeared in challenging water chemistries in our study. therefore, future work needs to establish the contribution to divergence of both extrinsic and intrinsic postzygotic isolation and the underlying genetic basis of both. such work will allow us to determine how postzygotic isolation evolves as a consequence of adaptation, the relative importance of extrinsic and intrinsic barriers, and how postzygotic isolation may act alone or in concert with prezygotic isolation to cause ecological speciation. | divergent natural selection has the potential to drive the evolution of reproductive isolation. the euryhaline killifish lucania parva has stable populations in both fresh water and salt water. lucania parva and its sister species, the freshwater l. goodei, are isolated by both prezygotic and postzygotic barriers. to further test whether adaptation to salinity has led to the evolution of these isolating barriers, we tested for incipient reproductive isolation within l. parva by crossing freshwater and saltwater populations. we found no evidence for prezygotic isolation, but reduced hybrid survival indicated that postzygotic isolation existed between l. parva populations. therefore, postzygotic isolation evolved before prezygotic isolation in these ecologically divergent populations. previous work on these species raised eggs with methylene blue, which acts as a fungicide. we found this fungicide distorts the pattern of postzygotic isolation by increasing fresh water survival in l. parva, masking species / population differences, and underestimating hybrid inviability. |
chemotherapy in patients with biliary tract cancer is indicated in those with unresectable advanced cancer and patients with recurrence after resection. however, no standard chemotherapy for biliary tract cancer has yet been established, because few randomized controlled trials (rcts) with large numbers of patients have been conducted to date. there are a number of studies regarding chemotherapy for biliary tract cancer, but many of these studies were prospective clinical trials with small numbers of patients, corresponding to phase ii studies, or retrospective studies, so high - level evidence in this area is limited. according to the classification of biliary tract carcinoma, bile duct cancer, gallbladder cancer, and ampullary cancer are classified as biliary tract cancer, and there are some clinical trials and articles in which treatment results that include those in intrahepatic bile duct cancer are also reported. if clinical trials are conducted for individual diseases separately, difficulties are encountered in view of efficiency and implementation, and so far, clinical trials of chemotherapy have been carried out only for biliary tract cancer in general. however, because treatment policy, sensitivity to chemotherapy, and prognosis differ widely from disease to disease, the treatment results of chemotherapy in biliary tract cancer should be evaluated on the basis of a full understanding of individual background factors. in the present guidelines, chemotherapy for unresectable biliary tract cancer and postoperative adjuvant chemotherapy has been described based on results from clinical trials and retrospective studies performed up to now. in order that effective chemotherapy for biliary tract cancer may be developed, active implementation of clinical trials is recommended. also, large multi - institutional rcts (phase iii) should be conducted to establish standard treatment as soon as possible. the only procedure in biliary cancer that confers a cure is curative surgical resection, so a diagnosis of incurability should be made with caution. lest the cancer be benign, surgical treatment should be administered as a rule only after pathological diagnosis (such as cytological and histological diagnoses) has been made. the chemotherapy described in the these guidelines is concerned with adenocarcinoma, which has the highest frequency of occurrence, so pertinent literature should be referred to in selecting treatment methods for other specific pathological types of cancer. in these guidelines, clinical questions (cqs) are posed, with responses in the form of recommendations (grades of the recommendations are defined in table 11). also, levels of evidence are given (in parentheses) for findings in reference citations (see definitions of levels in table 21). table 1strength of recommendationsa, strongly recommend performing the clinical actionb, recommend performing the clinical actionc1, the clinical action may be considered although there is a lack of high - level scientific evidence for its use. evidence insufficient to support or deny usefulnessd, recommend not performing the clinical actiontable 2levels of evidencelevel isystematic review / meta - analysislevel iione or more randomized clinical trialslevel iiinonrandomized controlled trialslevel ivanalytic epidemiology (cohort studies and case - control studies)level vdescriptive study (case reports and case - series studies)level viopinions of expert panels and individual experts not based on patient s data strength of recommendations chemotherapy is recommended in patients with good general physical condition (recommendation c1). to assess the efficacy of chemotherapy in achieving prolonged survival in biliary tract cancer, verification is necessary by comparing it with supportive treatment alone. to date, only two small rcts (level ii) have been published.2,3 an rct on chemotherapy and supportive treatment was conducted in patients with unresectable pancreas cancer and biliary tract cancer.2 in this study, fluorouracil (5-fu) + leucovorin or 5-fu + leucovorin + etoposide were used for chemotherapy. for all the patients, significantly prolonged survival was observed in the group who received chemotherapy (median survival time [mst ], 6.0 months) compared with the group who received supportive treatment alone (mst, 2.5 months). however, due to the small number of patients with biliary tract cancer (37 patients), no significant difference was established between the groups (chemotherapy group mst, 6.5 months ; supportive treatment group, 2.5 months ; p = 0.1). the rate of improvement in quality of life (qol) was also examined in this trial, and a significant difference was found in the chemotherapy group compared with the supportive treatment group (p < 0.01) ; an improvement of 36% was observed in the chemotherapy group (pancreas cancer, 38% ; biliary tract cancer, 33%) and an improvement of 10% (pancreas cancer, 13% ; biliary tract cancer, 5%) was shown in the supportive treatment group. the other small rct3 was conducted in japan, comparing chemotherapy with 5-fu + doxorubicin + mitomycin c (fam) and palliative treatment, such as bypass, in patients with unresectable pancreatic cancer, gallbladder cancer, and bile duct cancer. no significant improvement in prognosis was achieved in either group, but in the patients with gallbladder cancer, a good prognosis was achieved in the chemotherapy group (level ii). a retrospective analysis comparing chemotherapy and supportive treatment in patients with gallbladder cancer was carried out. no survival benefit owing to chemotherapy was observed in patients with a performance status of 2, while prolonged survival in the chemotherapy group was observed in patients with a performance status of 0 or 14 (level iv). concerning the efficacy of chemotherapy for unresectable biliary tract cancer, no evidence based on an rct with a large number of patients is available, but there are reports demonstrating the efficacy of chemotherapy in achieving improved qol and prolonged survival. thus, the strength of the recommendation for chemotherapy was determined as c1.24 the use of chemotherapy for biliary tract cancer should be limited to patients with unresectable, locally advanced disease and distant metastasis, or those with recurrence after resection. however, in patients with general deterioration (performance status of 2 or 3) or those with insufficient biliary decompression, the benefit of chemotherapy is small, so care should be taken in considering its indications. in these patients, palliative treatment targeting the maintenance of qol should be administered, including pain control and the placement of stents in the bile duct. as chemotherapy for unresectable advanced biliary tract carcinoma, gemcitabine or tegafur / gimeracil / oteracil potassium is recommended (recommendation c1). table 3 shows the results of systemic chemotherapy with a single agent for biliary tract cancer. the single use of fluoropyrimidines such as 5-fu, or a combination of 5-fu with interferon, leucovorin, or hydroxyurea as biochemical modulators, was often used for advanced biliary tract cancer. a favorable response rate of more than 30% has been reported with the combined use of a single agent with these modulators, but no difference was observed in mst, which ranged from 7 to 12 months, in studies in patients undergoing chemotherapy for unresectable biliary tract cancer, compared with patients who received best supportive care (level iii). in japan, uracil - tegafur (uft) is approved by the ministry of health, labor, and welfare for biliary tract cancer. however, regimens of uft alone or uft plus leucovorin were reported to have objective responses of 5% and 0%, respectively, and more than 60% patients were evaluated as having progressive disease in these regimen. therefore, uft should not be used alone for biliary tract cancer (level iii). table 3systemic chemotherapy with single agents for biliary tract canceragentnresponse ratemst (months)study designevidence levelauthoryearreferencefluorouracil5-fu180%rctlevel iitakada1994195-fu / lv / hu3030%8.0cohort studylevel iiigebbia1996225-fu/-ifn3234%12.0cohort studylevel iiipatt1996235-fu / lv1833%7.0cohort studylevel iiichen1998245-fu / lv2832%6.0cohort studylevel iiichoi2000255-fu / fa307%14.8cohort studylevel iiimalik200326uft / lv130%6.5cohort studylevel iiimani19995uft / lv160%4.5cohort studylevel iiichen20036capecitabine2619%cc, 8.1 ; gb, 9.9cohort studylevel iiipatt200427s-11921%8.3cohort studylevel iiiueno200428uft195%8.8cohort studylevel iiiikeda20057s-1400%9.4cohort studylevel iiifurusein press9taxanespaclitaxel150%cohort studylevel iiijones199629docetaxel160%cohort studylevel iiipazdur199930docetaxel2420%8.0cohort studylevel iiipapakostas200131gemcitabinegemcitabine (800 mg / m)3030%14.0cohort studylevel iiitsavaris200432gemcitabine (1000 mg / m)2536%7.0cohort studylevel iiigallardo200133gemcitabine (1000 mg / m)2413%7.2cohort studylevel iiilin200334gemcitabine (1000 mg / m)4018%7.6cohort studylevel iiiokusaka20068gemcitabine (1200 mg / m)196%6.5cohort studylevel iiiraderer199935gemcitabine (2200mg / m)150%4.6cohort studylevel iiieng200436gemcitabine (2200 mg / m)3222%11.5cohort studylevel iiipenz200127othersmitomycin c3010%4.5cohort studylevel iiitaal199338cisplatin138%5.5cohort studylevel iiiokada199439irinotecan368%6.1cohort studylevel iiialberts200240erlotinib428%7.5cohort studylevel iiiphilip200614mst, median survival time ; 5-fu, 5-fluorouracil ; ifn, interferon ; lv, levofolinic acid ; fa, folinic acid ; hu, hydroxyurea ; cc, cholangiocarcinoma ; gb gallbladder systemic chemotherapy with single agents for biliary tract cancer mst, median survival time ; 5-fu, 5-fluorouracil ; ifn, interferon ; lv, levofolinic acid ; fa, folinic acid ; hu, hydroxyurea ; cc, cholangiocarcinoma ; gb gallbladder in japan, none of the above modulators are approved by the ministry of health, labour, and welfare for use as chemotherapeutic drugs in biliary tract cancer. since 1999 although methods of administration are different, relatively good results are reported (table 3). a clinical trial (phase ii) was carried out with gemcitabine alone in japan.8 a response rate of 17.5% (95% confidence intervals [ci ], 7.3%32.8%), and an mst of 7.6 months was achieved with the standard dosage, which was 1000 mg / m as a 30-min intravenous infusion weekly, given for 3 consecutive weeks, followed by a week of rest, the results being roughly similar to those of reports from abroad. toxicity inducing myelosuppression, such as leucopenia, as well as nausea and anorexia, was mainly observed, but tolerance was good. based on these results, gemcitabine was approved by the ministry of health, labour, and welfare, in june, 2006, for use in biliary tract cancer (level iii). a clinical trial of tegafur / gimeracil / oteracil potassium (s-1), which is an oral anticancer drug that consists of tegafur (ft) as a prodrug of 5-fu, 5-chloro-2, 4-dihydroxypyridine (cdhp), and potassium oxonate (oxo), was conducted in japan. s-1 was orally administered at a dose of 80 mg / m per day for 28 days, followed by 14 days of rest. in a late phase ii trial, a favorable result was reported, with a success rate of 35% and mst of 9.4 months in 40 patients. because of this result, insurance coverage for the use of this agent for biliary tract cancer was endorsed in august, 2007 (level iii). there are also some reports of the use of mitomycin c, cisplatin, taxanes, and irinotecan (cpt-11) (table 3), but no satisfactory result has been achieved (level iii). for biliary tract cancer, there is a limitation in treatment effects brought about by chemotherapy with the use of a single agent, so many modalities of combination chemotherapy have been carried out (table 4). compared with single - agent chemotherapy, the response rate of combination chemotherapy is generally high and the survival period is also inclined to be long. although a regimen of a combination of 5-fu, anthracycline, and platinum has often been employed, no standard regimen has been established. an attempt at a regimen focusing on the use of gemcitabine is currently being made and a favorable result has been achieved, with the response rate being 21%48% and mst, 4.611.0 months in patients treated with gemcitabine + cisplatin1013 (level iii). currently, a large controlled trial comparing gemcitabine alone and gemcitabine + cisplatin (cddp) is being conducted chiefly by an english group and it is drawing attention. also, therapeutic drugs targeting molecular biological characteristics (molecular targeting therapy) are now under development. in view of a report suggesting the strong expression of epithelial growth factor receptor (egfr) in biliary tract cancer, a phase ii trial using erlotinib, which is an egfrinhibiting drug, is being carried out.14table 4combination chemotherapy for biliary tract cancerregimennresponse ratemst (months)study designevidence levelauthoryearreference5-fu - based5-fu / adm / mmc (fam)1429%8.5cohort studylevel iiiharvey198441epi / mtx/5-fu / lv170%9.0cohort studylevel iiikajanti1994425-fu / lv / mmc2025%9.5cohort studylevel iiiraderer199935mmc/5-fu / lv1926%6.0cohort studylevel iiichen200143platinum - basedepi / cddp/5-fu (ecf)2040%11.0cohort studylevel iiiellis199544cddp / epi/5-fu (cef)3719%5.9cohort studylevel iiimorizane2003455-fu / cddp2524%10.0cohort studylevel iiiducreux1998465-fu / cddp / lv2934%9.5cohort studylevel iiitaieb200247capecitabine / cddp4221%9.1cohort studylevel iiikim tw200348cddp / ifn / dxr/5-fu (piaf)3821%14.0cohort studylevel iiipatt200149epi / cddp / uft / lv4023%7.9cohort studylevel iiipark kh200550epi / cddp / capecitabine4340%8.0cohort studylevel iiipark sh2006515-fu / lv / carboplatin1421%5.0cohort studylevel iiisanz - altamira1998525-fu / lv / oxaliplatin (folfox)1619%9.5cohort studylevel iiinehls200253gemcitabine - basedgemcitabine / docetaxel439%11.0cohort studylevel iiikuhn200254gemcitabine/5-fu2733%5.3cohort studylevel iiiknox200455gemcitabine/5-fu / lv4212%4.7cohort studylevel iiihsu200456gemcitabine/5-fu / lv4212%9.7cohort studylevel iiialberts200557gemcitabine / cddp3038%4.6cohort studylevel iiidoval200410gemcitabine / cddp4028%8.4cohort studylevel iiithongprasert200511gemcitabine / cddp2935%11.0cohort studylevel iiikim st200612gemcitabine / cddp2733%10.0cohort studylevel iiipark bk200613gemcitabine / oxaliplatin3333%15.4cohort studylevel iiiandre200458gemcitabine / capecitabine4531%14.0cohort studylevel iiiknox200516gemcitabine / capecitabine4532%14.0cohort studylevel iiicho200559mst, median survival time ; mtx, methotrexate ; mmc, mitomycin c ; 5-fu, 5-fluorouracil ; lv, leucovorin ; ifn, interferon combination chemotherapy for biliary tract cancer mst, median survival time ; mtx, methotrexate ; mmc, mitomycin c ; 5-fu, 5-fluorouracil ; lv, leucovorin ; ifn, interferon in biliary tract cancer, treatment results differ widely depending upon the site of the cancer. in clinical trials, patients backgrounds, particularly the proportions of those with gallbladder cancer, bile duct cancer, and ampullary cancer, have a big impact on treatment results, such as the survival period.7,15,16 in a phase ii study of gemcitabine + capecitabine, the mst in patients with gallbladder cancer was 6.6 months and the mst in patients with bile duct cancer was 19 months ; thus, this difference in mst was assumed to be due to a biological difference.16 in overseas clinical trials of chemotherapy for biliary tract cancer, intrahepatic cholangiocarcinoma is often included. in japan, however, intrahepatic cholangiocarcinoma was often excluded from clinical trials of biliary tract cancer, because this entity is classified as primary liver cancer in the general rules for the clinical and pathological study of primary liver cancer,17 published by the liver cancer study group of japan18 and the tnm classification of malignant tumours, published by the international union against cancer. ideally, clinical trials of chemotherapy for biliary tract cancer and the assessment of treatment results should be carried out independently according to the individual diseases, but due to the small numbers of patients with the individual diseases, making an analysis of diseases independently is difficult. therefore, rcts with large numbers of patients and adequate stratification of all patients concerned should be conducted to evaluate the results of chemotherapy. the number of rcts that have been conducted concerning chemotherapy for bile duct cancer is not so large. outlined in table 5 are rcts retrieved from a literature search. in japan, comparisons were made by takada.3,19 between fam and 5-fu alone and between fam and palliative treatment, such as bypass operation. no significant difference in survival was confirmed in these rcts, and a standard chemotherapy has not been established yet (level ii). table 5randomized clinical trials for unresectable biliary tract cancernresponse ratemst (months) gb / bdp valuestudy designevidence levelauthor (year)reference1) oral 5-fu3010%4.9/6.1nsrctlevel iifalkson (1984)602) oral 5-fu + stz267.7%3.3/2.83) oral 5-fu + meccnu319.7%2.3/1.91) modified fam35 (18)4%6.2nsrctlevel iitakada (1994)192) 5-fu36 (18)0%6.01) 5-fu + lv or felv47 (18)11%6.5 (6.5)0.1rctlevel iiglimelius (1996)22) bsc43 (19)2.5 (2.5)1) modified fam145.2/4.1nsrctlevel iitakada (1998)32) palliative surgery172.4/7.71) mmc + gemcitabine2520%6.7rctlevel iikornek (2004)612) mmc + capecitabine2631%9.31) 5-fu295.0rctlevel iiducreux (2005)622) 5-fu + fa + cisplatin298.01) ecf279.00.72rctlevel iirao (2005)632) felv2712.0mst, median survival time ; gb, gallbladder ; bd, bile duct ; stz, streptozotocin ; meccnu, methyl - ccnu ; lv, levofolinic acid (lecovorin) ; bsc, best supportive care ; mmc, mitomycin c ; fa, folinic acid ; fam, 5-fu + adriamycin + mmc ; felv, 5-fu + etoposide + leucovorin ; ecf, epirubicin + cisplatin + 5-fu the total number of patients in the study, including those with pancreatic cancer ; numbers in parentheses, numbers of patients with biliary tract cancer the median overall survival time in all patients in the study, including those with pancreatic cancer ; numbers in parentheses, median overall survival times in patients with biliary tract cancer randomized clinical trials for unresectable biliary tract cancer mst, median survival time ; gb, gallbladder ; bd, bile duct ; stz, streptozotocin ; meccnu, methyl - ccnu ; lv, levofolinic acid (lecovorin) ; bsc, best supportive care ; mmc, mitomycin c ; fa, folinic acid ; fam, 5-fu + adriamycin + mmc ; felv, 5-fu + etoposide + leucovorin ; ecf, epirubicin + cisplatin + 5-fu the total number of patients in the study, including those with pancreatic cancer ; numbers in parentheses, numbers of patients with biliary tract cancer the median overall survival time in all patients in the study, including those with pancreatic cancer ; numbers in parentheses, median overall survival times in patients with biliary tract cancer as chemotherapy for biliary tract cancer at the present time, gemcitabine or s-1, which is covered by insurance in japan, is recommended on the basis of the results of many phase ii trials, including those performed in japan (level iii). evidence - based standard treatment should be established in the future by conducting controlled trials with the use of these agents. no recommendable regimen is available now, but it is hoped that adjuvant chemotherapy can be carried out as a clinical trial. early recurrence often occurs even if these patients have received curative resection ; the prognosis in patients with recurrence is extremely poor. therefore, further development of effective measures for preventing recurrence through postoperative adjuvant therapy is eagerly anticipated. in view of the low incidence of biliary tract cancer in western countries, where a large number of clinical studies of postoperative adjuvant therapy have been conducted in other types of cancer, few rcts of postoperative adjuvant therapy for biliary tract cancer have been carried out. on the other hand, in the east asian region (including japan), the incidence of biliary tract cancer is high so rcts of postoperative adjuvant therapy have been carried out in japan. from 1986 to 1992, takada. performed an rct in which 508 patients with pancreas cancer and biliary tract cancer were assigned to a group in which combination chemotherapy using 5-fu and mitomycin c (mmc) was administered (mf group) and a group for whom surgery alone was conducted (control group) ; the results were reported with a postoperative follow - up period of 5 years.20 as eligible patients, 158 patients with pancreatic cancer, 118 patients with bile duct cancer, 112 patients with gallbladder cancer, and 48 patients with ampullary cancer were chosen. analysis was done independently in the individual diseases, and the 5-year survival rate was found to be significantly better in the mf group for gallbladder cancer (table 6) (level ii). however, according to a review of the degree of cure due to resection, a significant difference in survival rate was found only in patients with noncurative resection. on the basis of an intent - to - treat analysis, no significant difference between the mf and the control group was observed in patients with gallbladder cancer. mf therapy has not yet been established as standard postoperative adjuvant therapy in biliary tract cancer, but the efficacy of postoperative adjuvant therapy is suggested by such trials. table 6randomized clinical trial of adjuvant chemotherapy for pancreas and biliary tract cancern5-year survival ratep valuepancreasmitomycin c/5-fu8111.5%nssurgery alone7718.0%gallbladdermitomycin c/5-fu6926.0%0.037surgery alone4314.4%biliary tractmitomycin c/5-fu5826.7%nssurgery alone6024.1%ampulla of vatermitomycin c/5-fu2428.1%nssurgery alone2434.3% randomized clinical trial of adjuvant chemotherapy for pancreas and biliary tract cancer todoroki summarized the results of clinical trials of chemotherapy for gallbladder cancer and verified that postoperative chemotherapy was efficacious. most of these clinical trials were conducted using a single arm, so no regimen could be selected as a possible candidate for postoperative adjuvant therapy.21 recently, chemotherapy focusing on gemcitabine for unresectable biliary tract cancer has been conducted, and regimens with good anticancer effects have sometimes been observed. however, no large rcts of postoperative adjuvant therapy have been carried out with the use of such regimens. rcts of postoperative adjuvant therapy, using new agents including gemcitabine and s-1, should be conducted vigorously. at the present time, no recommendable postoperative adjuvant therapy has been discovered, so clinical trials of various forms of chemotherapy are anticipated in the future. | few randomized controlled trials (rcts) with large numbers of patients have been conducted to date in patients with biliary tract cancer, and standard chemotherapy has not been established yet. in this article we review previous studies and clinical trials regarding chemotherapy for unresectable biliary tract cancer, and we present guidelines for the appropriate use of chemotherapy in patients with biliary tract cancer. according to an rct comparing chemotherapy and best supportive care for these patients, survival was significantly longer and quality of life was significantly better in the chemotherapy group than in the control group. thus, chemotherapy for patients with biliary tract cancer seems to be a significant treatment of choice. however, chemotherapy for patients with biliary tract cancer should be indicated for those with unresectable, locally advanced disease or distant metastasis, or for those with recurrence after resection. that is why making the diagnosis of unresectable disease should be done with greatest care. as a rule, pathological diagnosis, including cytology or histopathological diagnosis, is preferable. chemotherapy is recommended in patients with a good general condition, because in patients with general deterioration, such as those with a performance status of 2 or 3 or those with insufficient biliary decompression, the benefit of chemotherapy is limited. as chemotherapy for unresectable biliary tract cancer, the use of gemcitabine or tegafur / gimeracil / oteracil potassium is recommended. as postoperative adjuvant chemotherapy, no effective adjuvant therapy has been established at the present time. it is recommended that further clinical trials, especially large multi - institutional rcts (phase iii studies) using novel agents such as gemcitabine should be performed as soon as possible in order to establish a standard treatment. |
jaw bones and the associated soft tissues might be potential locations for different lesions, including cysts, inflammatory lesions and neoplasms with different radiographic features such as radiolucent, radiopaque and a mix of both ; these lesions might be located around the tooth root, around the crown and in the inter - radicular area and they might also have no relation with teeth (1). based on another classification, intraosseous lesions can be classified as inflammatory lesions, cysts, benign tumors, malignant neoplasms and osseous diseases found in the jaws and as the osseous manifestations of systemic conditions. differentiation of these lesions from the normal and anatomic structures of the jaws is of particular importance and since a dentist might be the first individual to diagnose these lesions, it is very important to have a proper knowledge of the prevalence and characteristics of these lesions for diagnosis and treatment planning (1, 2). retrospective studies are important for evaluation of geographic factors and variables such as gender, age and habits of individuals in a community as well as for estimation of the prevalence of such conditions in a community, because they can have a great role in identification of high - risk groups in the community and in the provision of preventive and therapeutic measures (3). in a study by becconsall - ryan and love in new zealand, 4983 radiolucent lesions related to odontogenic cysts (4), in a study by ali (5) a total of 385 developmental / inflammatory / reactive, cystic and tumor - like radiolucent lesions, in a study by acikgoz. (6), a total of 459 radiolucent lesions related to odontogenic and non - odontogenic cysts, and in a study by varkhede. (7), a total of 60 radiolucent lesions related to odontogenic tumors were reported. the majority of retrospective epidemiological studies have evaluated head and neck lesions in special categories of lesions such as malignant tumors, cysts or odontogenic tumors and some others have evaluated, in general, lesions in particular age groups. since sufficient data does not exist in the medical record of all the patients, including clinical history, laboratory tests, diagnostic procedures, the type of treatment and periodic follow - ups, it is necessary to carry out retrospective studies to collect the existing scattered data and transfer them to health care decision - making centers for proper programming. therefore, there is a paucity of studies that have evaluated the general pattern of different lesions in this area. in this context, there is no comprehensive study available in hamadan and thus, it is necessary to collect dispersed data on the subject in different diagnostic / treatment centers in hamadan province. the results of such retrospective studies can reveal differences in frequencies, the nature of the lesions and age distribution in comparison to other studies, helping designing and programming procedures in health care centers in every community, so that preventive therapeutic measures can be adopted. the aim of the present study was to evaluate the prevalence of intraosseous lesions in jaws as recorded in the files of patients referred to diagnostic and therapeutic centers in hamadan province, iran, during 1990 - 2010. in the present retrospective descriptive / cross - sectional study, the data sheets and radiographs of patients referring to public and private treatment centers in hamadan, consisting of 15 public centers and four private centers equipped with pathology laboratories, from 1998 to 2010, were reviewed by two reviewers. data sheets of the subjects were used to collect all the data of patients with intraosseous lesions, including age, gender, location of the lesion, the radiographic view of the lesions (radiolucent, radiopaque, mixed) and their types (inflammatory, cystic, benign and malignant neoplasms, manifestations of systemic conditions and osseous diseases of the jaws) and histopathological diagnosis. during this 20-year period, of 142865 samples, 287 intraosseous lesions had been recorded. in cases in which the data was not complete on the pathology report, the required data was completed using the patient s file in the department in which the biopsy had been taken and cases without a definitive diagnosis were excluded from the study. cases with more than one biopsy or subjects who did not have complete data such as site of lesion, gender, age, and pathological analysis were removed. the patients data were evaluated confidentially without using the patients names to consider ethical principles. data were analyzed with spss, using means, frequencies, standard deviations and relative frequencies separately for age, gender and location of the lesion. during the 20-year period, of 142865 samples, 287 intraosseous lesions had been recorded, 284 of which were included in the study and three were excluded because they did not have complete data. the mean age of the subjects was 28.8 15.2 years, with minimum and maximum ages of three and 72 years, respectively. table 2 presents the frequencies of different intraosseous lesions in terms of age and lesion type. table 3 presents the frequencies of different intraosseous lesions in terms of gender and lesion type. chi square = 56.4, degrees of freedom (df) = 8 and p value < 0.001 ; the relationship is significant. chi square = 25.1, degrees of freedom (df) = 24 and p value = 0.432 ; the relationship was not statistically significant. chi - squa r = 9.6, degrees of freedom (df) = 4 and p value = 0.045. the lesions were distributed in males and females almost similarly ; females = 48.8% and males = 51.2%. considering the type of lesions, the following frequency percentages were reported : 85.7% of inflammatory lesions in females ; 54.9% of manifestation of osseous diseases in jaws in females ; 54.8% of cystic lesions, 54.5% of benign tumors, and 57.9% of malignant lesions in males. a significant difference was observed between gender and type of lesion (p value = 0.045). regarding the frequencies of lesion locations, the lesions were found in the following locations in a descending order : posterior mandible (45.6%), anterior maxilla (38.1%), posterior maxilla (17.5%), and anterior mandible (8.8%). the majority of inflammatory lesions were located in the posterior mandible and maxilla (42.9%) ; cystic lesions and benign and malignant lesions of the jaws and manifestations of systemic conditions in the jaws were found in the posterior mandible with frequencies of 34.2%, 65.9%, 51.6% and 55.7%, respectively. a significant difference was observed between gender and type of lesion (p value 0.001). 287 intraosseous lesions were found among all the lesions evaluated, consisting of the following lesions in a descending order : cystic lesions (55.1%), manifestations of systemic conditions in jaws (18.1%), benign tumors (15.3%), malignant neoplasms (6.6%), and inflammatory lesions (9.4%). in a study by ali (5), the majority of lesions were cysts (55.1%), followed by inflammatory lesions (29.9%) and tumors (23.9%). the majority of intraosseous lesions in that study and in the present study were cysts, demonstrating consistency between the two studies. however, in the present study, manifestations of systemic conditions in the jaws were the second most prevalent lesions ; but, in the study by ali (5) inflammatory lesions were the second most prevalent lesions. such a disparity might be attributed to factors such as residential location, socioeconomic status, geographic location, and hygiene (5). in the present study, the majority of the lesions with radiolucent radiographic view were cysts, with radicular cysts, odontogenic keratocyst (okc) and dentigerous cysts being the most frequent ones. in a study by tortorici. (1) 1273 radiolucent lesions related to odontogenic cysts were reported, the most common of which were radicular cysts, okc and dentigerous cysts in a descending order. in the present study, 144 odontogenic cysts were reported, the majority of which had developed in males during the third decade of life in the maxilla. in studies by koseoglu. (9), prockt. (10), and acikgoz. (6), 90, 58, 680 and 452 cases of odontogenic cysts were reported, respectively. differences in the number of cases reported might be attributed to intervals, the number of patients referring to treatment factors, and some other factors. in the study by sanatkhani. (9), the majority of odontogenic cysts occurred during the second decade of life in contrast with the results of the present study. in the present study, 17.8% of the whole intraosseous lesions were radicular cysts, with a mean age of 33.3% years and an age range of 3 - 68 years. in addition, the majority of these lesions were found in males in the anterior maxilla with a radiolucent view. in studies by tortorici. (8) the majority of radicular cysts were reported in males ; however, in a study by jones. (11) a similar gender predilection was reported. in studies by jones. (6) the most common location for the radicular cysts was the anterior maxilla, consistent with the results of the present study. jones. (11) and prockt. (10) reported that these cysts developed most commonly in the third to fourth decades of life. reported that the majority of radicular cysts were radiolucent, consistent with the results of the present study (8). odontogenic keratocyst in the present study comprised 15.7% of all the intraosseous lesions, ranking the second in relation to prevalence. the mean age of the patients was 30.3% years and the majority of the lesions was located in the mandible with a radiolucent appearance and had a predilection for males. in the study by jones. (11) the prevalence of okc was higher in males, consistent with the results of the present study. (8) the prevalence of okc was equal in males and females, in contrary with our results. jones. (11), koseoglu. (8), and kondell and wiberg (12) reported that the most common location for okc was the posterior mandible, consistent with the results of the present study. the majority of okcs in the study by jones. (11) developed during the second and third decades of life. in the present study, the mean age of the patients was 29.5 years, consistent with the study above. in our study, dentigerous cysts comprised 9.1% of intraosseous lesions with a mean age of the patients being 27.1 years, with equal occurrence in males and females in the posterior mandible with a radiolucent view, ranking the third most common cyst. (11) reported a higher prevalence of dentigerous cysts in males ; however, prockt. (the results of the present study were in contrary with those of koseoglu. (8) and jones. (11) and consistent with those of prockt. in addition, consistent with the results of the present study, all the tree studies above reported a higher prevalence of dentigerous cysts in the mandible. since the majority of impacted teeth are mandibular third molars and such cysts are usually associated with impacted teeth, the most common site for these cysts is the posterior mandible (8, 10, 11). the mean age for the incidence of dentigerous cysts in the present study was the third decade of life, consistent with the mean age reported by koseoglu. (10) but not consistent with that reported by jones. (11) i.e., the fifth decade of life. in the present study, the majority of central giant cell granulomas were reported in males with a mean age of 26.8 years and with a radiolucent view in the posterior mandible. (13) reported that central giant cell granuloma was the second most common giant cell lesion after peripheral giant cell granuloma. if we do not consider the peripheral lesion, in the present study, central giant cell granuloma was the most common lesion among giant cell lesions, ie, cherubism, aneurysmal bone cysts, central giant cell granuloma and brown tumor, consistent with the study above. (13) reported that the prevalence of central giant cell granuloma was higher in females, which was different from the results of the present study ; however, the mean age of 23.72 years and the mandible were consistent with the results of the present study. in relation to fibro - osseous lesions, alsharif. (15), and worawongvasu and songkampol (16) reported that central cemento - ossifying fibroma and fibrous dysplasia were the most prevalent lesions, respectively, consistent with our results. (17) reported that the most common location for cemento - ossifying fibroma was the mandible, consistent with the results of the present study. the majority of these lesions were seen in females and males in studies by matsuzaka. ((15), respectively, inconsistent and consistent with the results of the present study, respectively. these lesions were most prevalent in the third and fourth decades of life in the study by worawongvasu and songkampol (16) and in the second decade of life in the study by matsuzaka. the majority of these lesions developed in the third decade of life, consistent and inconsistent with the results of the first and second studies above, respectively. the majority of these lesions were radiolucent in the present study, which was not consistent with the results of worawongvasu and songkampol (16) who reported that the majority of these lesions were mixed (16). in the studies carried out by worawongvasu and songkampol (16), and matsuzaka. (15), the majority of fibrous dysplasia lesions were seen in the mandible, maxilla, and with equal distribution between both arches, respectively. the results of the present study were consistent with those of matsuzaka. (17) and in contrary with those of the two other studies. the majority of these lesions were seen in the second decade of life in those three studies and in the third decade in the present study. in the studies by matsuzaka. (15), the majority of these lesions were seen in females and equal in but genders, respectively. the results of the present study were consistent with the first study and inconsistent with the latter. in the present study, the majority of these lesions were radiopaque, consistent with the results reported by worawongvasu and songkampol (16). (18), olgac. (19) and mamabol. (20) reported that the most common odontogenic tumor was ameloblastoma, consistent with the results of the present study. the majority of amelobastomas developed in the mandible in the present study, consistent with the results of the studies by olgac. (19), gunhan. (18), mamabolo. (20), and al - khateeb and ababneh (21) and varkhede. (7). based on the results of studies by al - khateeb and ababneh (21), varkhede. (20), ameloblastoma appeared most commonly in the third, third - fourth and fourth decades of life, respectively ; the results of the present study in this respect were consistent with the two first studies and inconsistent with those of the third study. however, chidzonga and mahomva reported a predilection for females (23). in the present study chidzonga and mahomva (23) and ajura and lau (22), reported that osteosarcoma was most prevalent at 27 and 7 - 68 years of age, respectively. in the present study, the mean age was 31.8 years, almost consistent with the first study above. the age range in the present study was 9 - 70 years, almost consistent with the second study. in the study carried out by matsuzaka. (17) the most common location for osteosarcomas was the mandible, consistent with the results of the present study. in the present study, 14 lesions were inflammatory in origin, with the most prevalent lesion being the periapical granuloma. the lesion had a predilection for females and the mean age was 32.8 years ; it was radiolucent and was most common in the mandible. in the studies by tortorici. (1) and stockdale and chandler (24), this lesion had a predilection for males and females, respectively. (1) the majority of lesions were identified in the third decade of life ; in the study by stockdale and chandler (24) the majority of lesions were diagnosed in the fourth decade, consistent with the results of the present study. in the present study, the majority of the lesions were identified in the mandible. in summary, apart from some similar cases, there were disparities with the results of previous studies. different factors such as residency, life style, socioeconomic status, geographic factors, racial factors, nutrition, hygiene, use of medications, presence of oral symptoms and signs, importance of biopsies based on the clinicians opinions or preparation of patients, referrals for sampling, and any other factors are the reasons for differences in the results. the results did not show the true prevalence of intraosseous lesions within the general population, but simply reected the frequency of histopathologically diagnosed lesions in an iranian population. this study evaluated the clinical variables such as gender and age in a community ; it can be used to estimate the prevalence of some conditions in the community and could have a great role in the provision of preventive and therapeutic measures ; but, since some data sheets of patient were not completed, some data were missed. therefore, we suggest that authorities complete records with accuracy, so that the researchers can conduct comprehensive studies and adopt some measures to prevent and treat many diseases. | background : jaw bones might be potential locations for different lesions. differences in prevalence and the type of lesions can help in designing and programming prevention procedures in health care centers.objectives:the aim of the present study was to evaluate the prevalence of intraosseous lesions in the jaws of patients referred to diagnostic and therapeutic centers in hamadan during 1990-2010.patients and methods : this cross - sectional descriptive analytical study was carried out in hamadan in 2011. data sheets of the subjects were used to collect all the data of patients with intraosseous lesions, including their age, gender, location of the lesion, the radiographic view of lesions, and their type and histopathological diagnoses. data were analyzed with spss, using means and frequencies.results:a total of 284 intraosseous lesions were reported in our study. the mean age of the subjects was 28.8 15.2 years. the lesions were distributed in males and females almost similarly. the most prevalent lesions were cystic lesions (54.58%), manifestations of systemic conditions in jaw bones (18.3%), benign tumors (15.5%), malignant lesions (6.7%), and inflammatory lesions (4.92%), in a descending order. the most common cystic lesion was radicular cyst ; the most common manifestation of systemic conditions in jaw bones was central giant cell granuloma ; the most common benign tumor was ameloblastoma ; the most common malignant lesion was osteosarcoma ; and the most common inflammatory lesion was periapical granuloma.conclusions:our data provided information on the prevalence and types of intraosseous lesions among an iranian population. this study provided baseline information to help in designing and programming procedures in health care centers in every community so that preventive therapeutic measures can be adopted. |
central nervous system (cns) tuberculosis commonly involves meninges, cerebrum, and cerebellum. an 18-year - old apparently normal unmarried female was admitted with complaints of sub acute onset fever, intermittent headache, neck pain, and altered behaviour for 10 days. she had no significant illness in the past and had normal menstrual cycles. on examination, her blood pressure was 110/80 mm of hg and had a regular pulse rate of 102 per min. contrast - enhanced magnetic resonance imaging (mri) of the brain showed a right thalamic infarct with multiple ring enhancing lesions in right parietal, occipital and pontine regions along with leptomeningeal enhancement [figure 1 ]. she was started on intravenous dexamethasone and oral antituberculosis treatment (att), which included oral isoniazid, rifampicin, pyrazinamide, and ethambutol. investigation profile on first and second admission (a) axial t2-weighted mri brain demonstrates high signal intensity infarct in right thalamus (white arrow). (b) sagittal post contrast t1-weighted mri brain demonstrates ring enhancing lesion (black arrow head) in pons and leptomeningeal enhancement (black arrow) she was readmitted five months later with complaints of weight gain, secondary amenorrhea for four months, apathy, excessive somnolence and intermittent headache for two weeks, and binocular diplopia for two days. on examination, her blood pressure was 110/80 mm of hg and had a regular pulse rate of 88 per min. a computed tomography (ct) scan of the brain showed hydrocephalus with cerebral edema and so an emergency ventriculo - peritoneal shunt was done [figure 2 ]. endocrinological investigation [table 2 ] revealed pituitary hypothyroidism, hypocortisolism, hypogonadotropic hypogonadism, and hyperprolactinemia. repeat contrast - enhanced mri of the brain revealed presence of exudates along basal cisterns and meningeal enhancement [figure 3 ]. (b) non contrast ct brain demonstrates in situ ventriculoperitoneal shunt tube hormonal profile on second admission sagittal post contrast t1-weighted magnetic resonance imaging brain demonstrates smaller pontine tuberculoma (black arrow head) and sellar suprasellar and leptomeningeal enhancement (black arrow) coronal post contrast t1-weighted magnetic resonance imaging brain demonstrates sellar suprasellar enhancement (white circle) she was restarted on oral att, hydrocortisone, thyroxine, cyclical estrogen, and progesterone. her condition dramatically improved up to an extent that she was now capable of carrying out her daily activities independently. she was discharged after rigorous counselling to remain compliant on att and hormone replacement therapy. our case is remarkable because it points to one of the very infrequently seen complication of neurological tuberculosis. hypothalamo - pituitary dysfunction due to tuberculosis can be due to the presence of a strategically placed tuberculoma or exudates around the sellar region. tubercle bacilli are believed to reach the pituitary by hematogenous spread from extra cranial sources or from the infection of skull base.[24 ] tuberculosis meningitis (tbm) has various radiological manifestations. contrast - enhanced mri of the brain usually shows leptomeningeal and basal cisternal enhancement, focal infarcts and tuberculomas usually surrounded by hypo attenuating edema (as seen in our case). the hydrocephalus is probably a result of adhesive meningeal reaction, obliteration of archnoid villi themselves or exudation in the subarachnoid space and cisterns of the base of the brain, around optic chiasma, interpeduncular, and prepontine cisterns. most of the available literature on pituitary tuberculosis is in the form of case reports. headache (91%), visual symptoms (46%), anterior pituitary hypo function (58%), hyperprolactinemia (23%), diabetes insipidus (11%) are the usual manifestations seen in a meta analysis of 54 cases of pituitary tuberculosis. our patient had hypogonadotropic hypogonadism and secondary hypothyroidism, which suggests the possibility of hypopitutarism. even when it was expected to be high in the face of tuberculosis (stress), the 8 a.m. serum fasting cortisol level was low. there was no visual field restriction, reduced visual acuity, or altered perception of red light. this suggests that there is probably no optic nerve involvement due to tuberculoma or exudation around optic chiasma. in pituitary tuberculosis, contrast - enhanced mri characteristically demonstrates thickening of the stalk due to chronic inflammatory scarring. the thickening of the stalk is non - specific and is described in diverse conditions like neoplasm, sarcoidosis, syphilis, lymphocytic hypophysitis, granulomatous hypophysitis, and eosinophilic granuloma.[135 ] the suprasellar extension can make evaluation of the stalk difficult on neuroimaging. the other mri findings described are peripheral ring enhancement of the mass, enhancement of the adjacent dura and basal enhancing exudates, sellar or suprasellar calcification, apoplexy, and erosion of the sellar floor. our patient had t2 weighted non homogenous hyper intensities, which enhanced on contrast in the sellar, suprasellar, and basal cisterns. although histopathological confirmation is emphasized by many authors, there are reports of pituitary tuberculosis that have been successfully managed on basis of clinical presentation and radiological findings. suprasellar tuberculosis presenting with pituitary - hypothalamic dysfunction can be differentiated from other neoplastic or inflammatory conditions on the basis of serial imaging findings, clinical profile, and response to att. when there is no real threat to the life or vision of the patient, then the major indication for a transphenoidal surgery would be for histopathological conformation. our patient had characteristic neuroimaging findings (tuberculomas, basal exudates, and leptomeningeal enhancement), hypoglycorrhachia and hyperproteinorrhachia suggestive of cns tuberculosis during both admissions. the fact that the pontine lesion regressed on att further asserts the diagnosis of cns tuberculosis [figure 3 ]. in the background of cns tuberculosis, noncompliance to treatment, the presence of hypothalamo - pituitary dysfunction, tuberculomas in sellar and suprasellar region almost confirms the diagnosis of pituitary tuberculosis.. however, there are reports that intracranial tuberculomas can develop or enlarge during antituberculous therapy and exaggerated host reaction to tuberculous protein is thought to play a role. this could probably explain the normal pituitary imaging during the first admission in our patient. however, because hormone analysis was not done during the first admission, it is not possible to rule out asymptomatic hypopituitarism. there is no standardized regimen regarding the drugs, dosages, use of steroids, and duration of treatment for cns tuberculosis. treatment is individualized in most of the cases. as the experience with tuberculomas of pituitary our patient was treated with a regimen that is usually followed for cns tuberculosis and hormone replacement therapy. | neurological tuberculosis can very rarely involve the hypophysis cerebri. we report a case of an eighteen year old female who presented with five months duration of generalised apathy, secondary amenorrhea and weight gain. she was on irregular treatment for tuberculosis of the central nervous system for the last five months. neuroimaging revealed sellar and suprasellar tuberculomas and communicating hydrocephalus requiring emergency decompression. endocrinological investigation showed hypopituitarism manifesting as pituitary hypothyroidism, hypocortisolism, hypogonadotropic hypogonadism, and hyperprolactinemia. restarting anti - tuberculosis treatment, hormone replacement therapy, and a ventriculo - peritoneal shunt surgery led to remarkable improvement in the general condition of the patient. |
this report reflects the consensus of the clinical and translational science awards (ctsa) consortium enhancing clinical research professionals training and qualification (ecrptq) project that responded to a mandate to improve the efficiency of clinical trials through educating principal investigators (pis) and clinical research coordinators (crcs) in core clinical trial competencies. the objectives of this project were to codify the core competencies into a single high - level set of standards that could serve as the framework for defining professional competency across the clinical research continuum. there is pressure to provide institution - specific training for some aspects of job performance, but we believe there is significant value and efficiency to be gained by a uniform curriculum that trains to a standard set of competencies. the institute of medicine (iom) 2012 workshop report entitled envisioning a transformed clinical trials enterprise in the united states, stated that the more traditional areas of mechanistic research and efficacy trials call for specialized workforces that until now have all too often depended on ad hoc, on - the - job learning as opposed to the prospective training and education that defines a mature discipline. the data from the us food and drug administration center for drug evaluation and research inspections from fiscal years 20042011 (n=2325) of us clinical trial sites show the following clinical investigator deficiencies : 42% protocol violations, 30% record - keeping deficiencies, 12% informed consent deficiencies, 10% of drug accountability violations, 9% institutional review board (irb) communication, and 5% with problems reporting adverse events (aes). moreover, despite recent requirements for good clinical practice (gcp) training of investigators and clinical research staff, a decrease in these deficiencies and violations have persisted (see http://bit.ly/1mali8w). these data suggest that there is a need for an intervention to increase clinical investigator and coordinator competence and improve clinical trial performance metrics. we hypothesize that clinical trial (and particularly multisite clinical trial) performance will be significantly improved by a standard set of systematically harmonized competencies that equip pis and crcs with the necessary skills to more effectively, efficiently, and safely execute clinical trials. the iom has issued a challenge to create a clinical research workforce that can address increasing complexity in clinical and translational research and generate study results that reach the community more efficiently. this challenge has focused attention on a clinical research workforce that includes not only pis, but also crcs and staff members. to date, education and training for pis has evolved through the ctsa, but this training did not reach all investigators, nor the many clinical research professionals, and other team members who were external to the local ctsa funding mechanism. mandates for gcp training by industry sponsors and irbs have provided a minimal training activity for investigators and their clinical research teams. academic and nonacademic sites provide local training and educational links, but often these efforts are unfunded and/or institution - specific activities. a formalized education and training requirement is lacking for individuals working in this profession. over the past decade, various professional societies and institutes have supported a cbe approach for clinical research professionals. cbe identifies specific learning outcomes for knowledge and its application and is often referred to as outcomes - based education. part of an educational trend that emerged in the 1970s, this approach has been endorsed by the iom in its 2005 report. characteristics that distinguish cbe include the following:learner outcomes that are based on analysis of typical job responsibilities of practitioners.a curriculum focused on what learners need to learn to perform specific job tasks, not necessarily on traditional subject matter.hierarchically sequenced modules that allow learners to proceed at their own pace.educators employing assessment techniques that measure learner performance in settings that approximate the real environment. a curriculum focused on what learners need to learn to perform specific job tasks, not necessarily on traditional subject matter. educators employing assessment techniques that measure learner performance in settings that approximate the real environment. the evolution of cbe / competency - based training and the publication of core competencies in clinical research offer a pathway for achieving workforce development goals. cbe / competency - based training promise a skilled workforce to a variety of stakeholders by mapping core competencies to educational and training curricula. the competency - based approach ultimately defines competences and qualifications in a systematic learner - centric pathway. competencies represent not only basic knowledge, but higher levels of knowledge, skills, and attitudes (ksas) that embody the profession. core competencies for clinical research nurse coordinators evolved from several works generated by the royal college of nursing in the united kingdom, oncology nursing society, and a national institutes of health (nih) clinical research nurse working group [79 ]. other role delineation work for clinical research nurses have continued to study these domains and specific ksas in practice [1013 ]. many of the role delineations attributed to clinical research nurses can also be attributed to non - nurse crcs. in addition, core competencies for clinical and translational pis were featured on the ctsa web site and formed the basis for curriculum development for master - level courses under the ctsa, beginning with the k-30 awards. the role of pharmaceutical physicians, which is primarily a role found in europe and south america, the iom has issued a challenge to create a clinical research workforce that can address increasing complexity in clinical and translational research and generate study results that reach the community more efficiently. this challenge has focused attention on a clinical research workforce that includes not only pis, but also crcs and staff members. to date, education and training for pis has evolved through the ctsa, but this training did not reach all investigators, nor the many clinical research professionals, and other team members who were external to the local ctsa funding mechanism. mandates for gcp training by industry sponsors and irbs have provided a minimal training activity for investigators and their clinical research teams. academic and nonacademic sites provide local training and educational links, but often these efforts are unfunded and/or institution - specific activities. over the past decade, various professional societies and institutes have supported a cbe approach for clinical research professionals. cbe identifies specific learning outcomes for knowledge and its application and is often referred to as outcomes - based education. part of an educational trend that emerged in the 1970s, this approach has been endorsed by the iom in its 2005 report. characteristics that distinguish cbe include the following:learner outcomes that are based on analysis of typical job responsibilities of practitioners.a curriculum focused on what learners need to learn to perform specific job tasks, not necessarily on traditional subject matter.hierarchically sequenced modules that allow learners to proceed at their own pace.educators employing assessment techniques that measure learner performance in settings that approximate the real environment. a curriculum focused on what learners need to learn to perform specific job tasks, not necessarily on traditional subject matter. educators employing assessment techniques that measure learner performance in settings that approximate the real environment. the evolution of cbe / competency - based training and the publication of core competencies in clinical research offer a pathway for achieving workforce development goals. cbe / competency - based training promise a skilled workforce to a variety of stakeholders by mapping core competencies to educational and training curricula. the competency - based approach ultimately defines competences and qualifications in a systematic learner - centric pathway. competencies represent not only basic knowledge, but higher levels of knowledge, skills, and attitudes (ksas) that embody the profession. core competencies for clinical research nurse coordinators evolved from several works generated by the royal college of nursing in the united kingdom, oncology nursing society, and a national institutes of health (nih) clinical research nurse working group [79 ]. other role delineation work for clinical research nurses have continued to study these domains and specific ksas in practice [1013 ]. many of the role delineations attributed to clinical research nurses can also be attributed to non - nurse crcs. in addition, core competencies for clinical and translational pis were featured on the ctsa web site and formed the basis for curriculum development for master - level courses under the ctsa, beginning with the k-30 awards. the role of pharmaceutical physicians, which is primarily a role found in europe and south america, the ecrptq leadership team considered a variety of competency frameworks for this phase:the ctsa education and career development key function committee developed the ctsa master - level competencies, approved in 2011 (http://bit.ly/21cx5n8), to define the training standards for individuals functioning at the master s level in clinical and translational research. this framework includes 14 thematic areas that are intended to shape the training experiences of early career investigators and it represents the foundation for many graduate programs in clinical research across the ctsa consortium. although the ecrptq leadership team felt these competencies to be highly relevant for investigators, they did not fully address the necessary qualifications and skills for other team members. the ecrptq leadership team also examined specialty competencies in a variety of areas developed by ctsa key function committees, including bioinformatics.the nih clinical research nursing domains of practice for the specialty of clinical research nursing (http://cc.nih.gov/nursing/crn/dop_document.pdf) was another framework that was reviewed and considered, but also did not necessarily address the qualifications and skills for all team members.competencies outlined by the oncology nursing society (https://www.ons.org/sites/default/files/ctncompetencies.pdf) were also reviewed but the group felt they were rather limited, focusing only on oncology studies.the ecrptq leadership also acknowledged the work of the national research coordinator consortium, formerly known as the ctsa research coordinator taskforce. like the other domains listed previously, the ecrptq leadership felt a framework that would be inclusive of all study team members was needed. the ctsa education and career development key function committee developed the ctsa master - level competencies, approved in 2011 (http://bit.ly/21cx5n8), to define the training standards for individuals functioning at the master s level in clinical and translational research. this framework includes 14 thematic areas that are intended to shape the training experiences of early career investigators and it represents the foundation for many graduate programs in clinical research across the ctsa consortium. although the ecrptq leadership team felt these competencies to be highly relevant for investigators, they did not fully address the necessary qualifications and skills for other team members. the ecrptq leadership team also examined specialty competencies in a variety of areas developed by ctsa key function committees, including bioinformatics. the nih clinical research nursing domains of practice for the specialty of clinical research nursing (http://cc.nih.gov/nursing/crn/dop_document.pdf) was another framework that was reviewed and considered, but also did not necessarily address the qualifications and skills for all team members. competencies outlined by the oncology nursing society (https://www.ons.org/sites/default/files/ctncompetencies.pdf) were also reviewed but the group felt they were rather limited, focusing only on oncology studies. the ecrptq leadership also acknowledged the work of the national research coordinator consortium, formerly known as the ctsa research coordinator taskforce. like the other domains listed previously, the ecrptq leadership felt a framework that would be inclusive of all study team members was needed. ultimately, the framework proposed by the joint task force for clinical trial competency (jtf) was selected that identified 8 broad domains of competence:(1)scientific concepts and research design(2)ethical and participant safety considerations(3)medicines development and regulation(4)clinical trial operations(5)study and site management(6)data management and informatics(7)leadership and professionalism(8)communication and teamwork scientific concepts and research design ethical and participant safety considerations medicines development and regulation clinical trial operations study and site management data management and informatics leadership and professionalism communication and teamwork the ecrptq leadership team selected this framework because of its comprehensive applicability and its widespread uptake by numerous other stakeholders in the clinical trial enterprise. an additional consideration was the knowledge that the jtf mapped its competency framework to those mentioned previously, along with additional competency frameworks identified by consortium of academic programs in clinical research (coapcr) and the uk national health service. the jtf model is displayed in table 1.table 1jtf core competenciesdomaindefinitioncompetenciesscientific concepts and research designknowledge of scientific concepts related to the design and analyses of clinical trials5ethical and participant safety considerationsknowledge of the care of patients, human subject protections, and safety in the conduct of a clinical trial8medicines development and regulationknowledge of how drugs, biologics, and devices are developed and regulated7clinical trial operationsknowledge of study management, gcp compliance (regulatory affairs), safety reporting (adverse event identification and reporting, postmarket surveillance, pharmacovigilance), and the handling of investigational product12study and site managementknowledge of requirements for site management (financial, personnel, including site and study operations, not including regulatory affairs)6data management and informaticsknowledge of how data are acquired and managed during a clinical trial (source data, data entry, queries, quality control, corrections) and the concept of a locked database5leadership and professionalismknowledge of the principles and practice of leadership and professionalism in clinical research4communication and teamworkknowledge of all elements of communication within the site and between the site and sponsors, contract research organizations, regulators. this ctsa - wide endeavor drew upon expertise across the consortium for this important and complex project. the ecrptq leadership team invited members of the ctsa consortium with relevant expertise to participate in each of the competency domain working groups, which were created based on the 8 competency domains identified by the jtf. the expectations of the groups included a desire to focus on 2 roles : pis and crcs conducting clinical trials. the deliverables also included the charge to review and refine the jtf competency statements, identify assessment areas for each competency statement, and determine gaps in existing training. as part of the first phase of the ecrptq project, a social / behavioral research (s / br) working group was created in response to a recognized need to address gcp in an appropriate and meaningful way for researchers conducting clinical trial testing behavioral interventions. this work is described in a separate paper authored by murphy and her colleagues in this journal. in this phase of the ecrptq project, members of the s / br working group were invited to participate in each of the competency domain working groups, contributing their expertise. workgroups began working as soon as co - leads were identified and workgroup membership assigned. s / br working group members were embedded within the competency domain working groups to provide feedback to ensure that the competencies were inclusive of s / br. after a series of conference calls, email exchanges, and 2 working meetings in 2015, all competency domain working groups submitted their deliverables to the leadership team. following this submission, a review team comprised of individuals from across the consortium conducted a thorough appraisal of this work to synthesize and collate the materials and provide a final draft to be reviewed by the project leadership team before being forwarded to the ctsa steering committee. the core competencies were then reviewed by the jtf, coapcr, and association of clinical research professionals (acrp). the final meeting was attended by project leadership, the competency domain co - leads, jtf, and coapcr. at the final meeting, the attendees revisited the importance of focusing on drafting competency statements that represent clear and measurable expressions of performance for professionals involved in clinical trials. the attendees also discussed areas of potential overlap within the framework, with some competencies appearing in more than 1 general competency domain. the 51 competency statements written by the jtf were carefully reviewed by competency domain working groups and review teams. of the 51 jtf statements, 34 were modified to enhance meaning and to reflect a focus on clinical trials. five of the jtf competency statements were removed as stand - alone statements and were rewritten as assessments for other competencies, and 3 new ecrptq competency statements were added. in total, 48 ecrptq competency statements reflect the work of these groups (see appendix 1). several items are particularly noteworthy regarding the overall work:first, the competency domain working group reviewing the medicines development and regulation domain called for a renaming of that domain to investigational products development and regulation to be more inclusive of device research.second, the domain communication and teamwork was separated into 2 domains at the request of the ecrptq leadership team early in the phase ii process, believing the concept of team science to be of critical importance for the ctsa consortium. because the field of team science is still emerging and that the relevant skills are still being defined, the decision was made that the domain should be combined with the leadership and professionalism domain to become leadership, professionalism, and team science. as team science competencies emerge and are refined, it may make sense later to separate the domains.third, there are purposefully some areas of overlap across domains, as the group agreed that understanding concepts can be very related but still highly nuanced.the s / br working group suggested additional edits to 6 competency statements in 3 competency domains. these suggestions are relevant to study teams specifically conducting clinical trials involving behavioral interventions or assessments.echoing a sentiment expressed by the jtf at its meeting in april 2015, the group recognized the importance of regular updates and revisions to this work, corresponding to advances in science and concomitant regulations. first, the competency domain working group reviewing the medicines development and regulation domain called for a renaming of that domain to investigational products development and regulation to be more inclusive of device research. second, the domain communication and teamwork was separated into 2 domains at the request of the ecrptq leadership team early in the phase ii process, believing the concept of team science to be of critical importance for the ctsa consortium. because the field of team science is still emerging and that the relevant skills are still being defined, the decision was made that the domain should be combined with the leadership and professionalism domain to become leadership, professionalism, and team science. as team science competencies emerge and are refined, it may make sense later to separate the domains. third, there are purposefully some areas of overlap across domains, as the group agreed that understanding concepts can be very related but still highly nuanced. the s / br working group suggested additional edits to 6 competency statements in 3 competency domains. these suggestions are relevant to study teams specifically conducting clinical trials involving behavioral interventions or assessments. echoing a sentiment expressed by the jtf at its meeting in april 2015, the group recognized the importance of regular updates and revisions to this work, corresponding to advances in science and concomitant regulations. competency statements are broad and meant to be generally applicable to both pis and crcs ; however, in thinking of how to apply these statements to different roles within a study team, competency domain working groups identified areas of assessment specific to investigators and crcs, with a principal focus on entry - level individuals. competency domain working groups were provided with an overview of bloom s taxonomy to assist them in writing measurable and appropriately leveled assessments that are at higher level ksas. most assessments proposed by the competency domain working groups are specific and measurable, such as prepare a research question and demonstrate knowledge of appropriate control, storage, and dispensing of investigational products. groups identified a total of 429 potential assessments across all domains, with 220 identified as appropriate for investigators and 209 identified as appropriate for crcs, a list that is in no way exhaustive. a considerable number of these assessments are identical for investigators and for crcs, so the total does not represent unique assessments. in terms of methods, many competency domain working groups suggested the use of case studies and observation of behavior, indicating that simply passing a quiz or multiple - choice exam is not indicative of mastery ; some also recommended pre / post testing. it is important to acknowledge that while the creation of detailed assessment methods was not within the scope of this work, some working groups did suggest methods that might be employed. competency domain working groups were also tasked with identifying and examining existing training believed to address the competencies identified within the groups respective competency domains. they took a broad approach to this task, based on the expertise and experiential knowledge of their members. it was not possible to examine every training program that might exist, but the groups reviewed a reasonably representative sample of offerings. in general, included were training and education offered by ctsa institutions ; by professional organizations devoted to education and resources ; by industry ; and by government units (see http://bit.ly/1iv821x, entitled training gaps by competency domains). collectively, competency domain working groups examined the following number of education and training offerings (with some offerings identified as appropriate for multiple competencies) (see table 2).table 2summary of existing education and trainingsctsa training and educationprofessional organizations education and trainingindustry education and traininggovernment education and training132140863total=343 offerings (not unique)ctsa, clinical and translational science awards. from table 2, of the 343, 219 unique education and training offerings were identified. every competency domain had associated training with a few exceptions, noted below: leadership, professionalism, and team science : identify and apply professional guidelines and codes of ethics as they relate to the conduct of clinical trials. no training identified. leadership, professionalism, and team science : describe the methods necessary to work effectively with multidisciplinary and interprofessional research teams. no training identified. communication : describe the component parts of a traditional scientific publication. leadership, professionalism, and team science : identify and apply professional guidelines and codes of ethics as they relate to the conduct of clinical trials. leadership, professionalism, and team science : describe the methods necessary to work effectively with multidisciplinary and interprofessional research teams. appear to be available online ; not all of those offerings are readily accessible in the public domain, or free of charge, however. in addition to the trainings identified as meeting - specified competencies, groups cited 214 supplemental resources, including web sites, reports, books, and published articles. see http://bit.ly/1u3pybs for the catalog of identified existing education and trainings organized by competency domain. sample online offerings:northwestern university, clinical and translational sciences institute introduction to clinical research online modulesuniversity of washington, institute of translational health sciences (iths), self - directed learning centeroffice of research integrity : the lab, the research clinic nih : teaching the responsible conduct of research acrp : gcp an introduction to ich gcp guidelines collaborative institutional training initiative (citi) : populations in research requiring additional consideration uc davis : strengthening provider patient communication skills in clinical trials. northwestern university, clinical and translational sciences institute introduction to clinical research online modules university of washington, institute of translational health sciences (iths), self - directed learning center office of research integrity : the lab, the research clinic nih : teaching the responsible conduct of research acrp : gcp an introduction to ich gcp guidelines collaborative institutional training initiative (citi) : populations in research requiring additional consideration uc davis : strengthening provider patient communication skills in clinical trials. competency domain working groups were not tasked to consider cost as a factor in the groups work, but it is certainly an important consideration and some groups did so. the cost of these offerings ranges from free to a significant financial investment by the department or individual. similarly, the working groups were not tasked to consider training quality, but many did so nevertheless identifying the need for training that actively engages the learner, going beyond mere rote memorization. a number of competency domain working groups noted that existing training focuses too heavily on theoretical concepts and historical events and not enough on the application of knowledge. competency domain working groups frequently noted that online offerings are insufficient alone and must be supplemented by local institutional education and training. the education and training offerings that are listed in this report (see http://bit.ly/1u3pybs) are not endorsed by the ecrptq leadership team. it was outside the parameters of this project to conduct a thorough evaluation of the suggested trainings from the competency domain working groups. there was broad agreement that the assessment of their quality should be undertaken as a future phase of the project. the ecrptq leadership team considered a variety of competency frameworks for this phase:the ctsa education and career development key function committee developed the ctsa master - level competencies, approved in 2011 (http://bit.ly/21cx5n8), to define the training standards for individuals functioning at the master s level in clinical and translational research. this framework includes 14 thematic areas that are intended to shape the training experiences of early career investigators and it represents the foundation for many graduate programs in clinical research across the ctsa consortium. although the ecrptq leadership team felt these competencies to be highly relevant for investigators, they did not fully address the necessary qualifications and skills for other team members. the ecrptq leadership team also examined specialty competencies in a variety of areas developed by ctsa key function committees, including bioinformatics.the nih clinical research nursing domains of practice for the specialty of clinical research nursing (http://cc.nih.gov/nursing/crn/dop_document.pdf) was another framework that was reviewed and considered, but also did not necessarily address the qualifications and skills for all team members.competencies outlined by the oncology nursing society (https://www.ons.org/sites/default/files/ctncompetencies.pdf) were also reviewed but the group felt they were rather limited, focusing only on oncology studies.the ecrptq leadership also acknowledged the work of the national research coordinator consortium, formerly known as the ctsa research coordinator taskforce. like the other domains listed previously, the ecrptq leadership felt a framework that would be inclusive of all study team members was needed. the ctsa education and career development key function committee developed the ctsa master - level competencies, approved in 2011 (http://bit.ly/21cx5n8), to define the training standards for individuals functioning at the master s level in clinical and translational research. this framework includes 14 thematic areas that are intended to shape the training experiences of early career investigators and it represents the foundation for many graduate programs in clinical research across the ctsa consortium. although the ecrptq leadership team felt these competencies to be highly relevant for investigators, they did not fully address the necessary qualifications and skills for other team members. the ecrptq leadership team also examined specialty competencies in a variety of areas developed by ctsa key function committees, including bioinformatics. the nih clinical research nursing domains of practice for the specialty of clinical research nursing (http://cc.nih.gov/nursing/crn/dop_document.pdf) was another framework that was reviewed and considered, but also did not necessarily address the qualifications and skills for all team members. competencies outlined by the oncology nursing society (https://www.ons.org/sites/default/files/ctncompetencies.pdf) were also reviewed but the group felt they were rather limited, focusing only on oncology studies. the ecrptq leadership also acknowledged the work of the national research coordinator consortium, formerly known as the ctsa research coordinator taskforce. like the other domains listed previously, the ecrptq leadership felt a framework that would be inclusive of all study team members was needed. ultimately, the framework proposed by the joint task force for clinical trial competency (jtf) was selected that identified 8 broad domains of competence:(1)scientific concepts and research design(2)ethical and participant safety considerations(3)medicines development and regulation(4)clinical trial operations(5)study and site management(6)data management and informatics(7)leadership and professionalism(8)communication and teamwork scientific concepts and research design ethical and participant safety considerations medicines development and regulation clinical trial operations study and site management data management and informatics leadership and professionalism communication and teamwork the ecrptq leadership team selected this framework because of its comprehensive applicability and its widespread uptake by numerous other stakeholders in the clinical trial enterprise. an additional consideration was the knowledge that the jtf mapped its competency framework to those mentioned previously, along with additional competency frameworks identified by consortium of academic programs in clinical research (coapcr) and the uk national health service. the jtf model is displayed in table 1.table 1jtf core competenciesdomaindefinitioncompetenciesscientific concepts and research designknowledge of scientific concepts related to the design and analyses of clinical trials5ethical and participant safety considerationsknowledge of the care of patients, human subject protections, and safety in the conduct of a clinical trial8medicines development and regulationknowledge of how drugs, biologics, and devices are developed and regulated7clinical trial operationsknowledge of study management, gcp compliance (regulatory affairs), safety reporting (adverse event identification and reporting, postmarket surveillance, pharmacovigilance), and the handling of investigational product12study and site managementknowledge of requirements for site management (financial, personnel, including site and study operations, not including regulatory affairs)6data management and informaticsknowledge of how data are acquired and managed during a clinical trial (source data, data entry, queries, quality control, corrections) and the concept of a locked database5leadership and professionalismknowledge of the principles and practice of leadership and professionalism in clinical research4communication and teamworkknowledge of all elements of communication within the site and between the site and sponsors, contract research organizations, regulators. this ctsa - wide endeavor drew upon expertise across the consortium for this important and complex project. the ecrptq leadership team invited members of the ctsa consortium with relevant expertise to participate in each of the competency domain working groups, which were created based on the 8 competency domains identified by the jtf. the expectations of the groups included a desire to focus on 2 roles : pis and crcs conducting clinical trials. the deliverables also included the charge to review and refine the jtf competency statements, identify assessment areas for each competency statement, and determine gaps in existing training. as part of the first phase of the ecrptq project, a social / behavioral research (s / br) working group was created in response to a recognized need to address gcp in an appropriate and meaningful way for researchers conducting clinical trial testing behavioral interventions. this work is described in a separate paper authored by murphy and her colleagues in this journal. in this phase of the ecrptq project, members of the s / br working group were invited to participate in each of the competency domain working groups, contributing their expertise. workgroups began working as soon as co - leads were identified and workgroup membership assigned. s / br working group members were embedded within the competency domain working groups to provide feedback to ensure that the competencies were inclusive of s / br. after a series of conference calls, email exchanges, and 2 working meetings in 2015, all competency domain working groups submitted their deliverables to the leadership team. following this submission, a review team comprised of individuals from across the consortium conducted a thorough appraisal of this work to synthesize and collate the materials and provide a final draft to be reviewed by the project leadership team before being forwarded to the ctsa steering committee. the core competencies were then reviewed by the jtf, coapcr, and association of clinical research professionals (acrp). the final meeting was attended by project leadership, the competency domain co - leads, jtf, and coapcr. at the final meeting, the attendees revisited the importance of focusing on drafting competency statements that represent clear and measurable expressions of performance for professionals involved in clinical trials. the attendees also discussed areas of potential overlap within the framework, with some competencies appearing in more than 1 general competency domain. the 51 competency statements written by the jtf were carefully reviewed by competency domain working groups and review teams. of the 51 jtf statements, 34 five of the jtf competency statements were removed as stand - alone statements and were rewritten as assessments for other competencies, and 3 new ecrptq competency statements were added. in total, 48 ecrptq competency statements reflect the work of these groups (see appendix 1). several items are particularly noteworthy regarding the overall work:first, the competency domain working group reviewing the medicines development and regulation domain called for a renaming of that domain to investigational products development and regulation to be more inclusive of device research.second, the domain communication and teamwork was separated into 2 domains at the request of the ecrptq leadership team early in the phase ii process, believing the concept of team science to be of critical importance for the ctsa consortium. because the field of team science is still emerging and that the relevant skills are still being defined, the decision was made that the domain should be combined with the leadership and professionalism domain to become leadership, professionalism, and team science. as team science competencies emerge and are refined, it may make sense later to separate the domains.third, there are purposefully some areas of overlap across domains, as the group agreed that understanding concepts can be very related but still highly nuanced.the s / br working group suggested additional edits to 6 competency statements in 3 competency domains. these suggestions are relevant to study teams specifically conducting clinical trials involving behavioral interventions or assessments.echoing a sentiment expressed by the jtf at its meeting in april 2015, the group recognized the importance of regular updates and revisions to this work, corresponding to advances in science and concomitant regulations. first, the competency domain working group reviewing the medicines development and regulation domain called for a renaming of that domain to investigational products development and regulation to be more inclusive of device research. second, the domain communication and teamwork was separated into 2 domains at the request of the ecrptq leadership team early in the phase ii process, believing the concept of team science to be of critical importance for the ctsa consortium. because the field of team science is still emerging and that the relevant skills are still being defined, the decision was made that the domain should be combined with the leadership and professionalism domain to become leadership, professionalism, and team science. as team science competencies emerge and are refined, it may make sense later to separate the domains. third, there are purposefully some areas of overlap across domains, as the group agreed that understanding concepts can be very related but still highly nuanced. the s / br working group suggested additional edits to 6 competency statements in 3 competency domains. these suggestions are relevant to study teams specifically conducting clinical trials involving behavioral interventions or assessments. echoing a sentiment expressed by the jtf at its meeting in april 2015, the group recognized the importance of regular updates and revisions to this work, corresponding to advances in science and concomitant regulations. competency statements are broad and meant to be generally applicable to both pis and crcs ; however, in thinking of how to apply these statements to different roles within a study team, competency domain working groups identified areas of assessment specific to investigators and crcs, with a principal focus on entry - level individuals. competency domain working groups were provided with an overview of bloom s taxonomy to assist them in writing measurable and appropriately leveled assessments that are at higher level ksas. most assessments proposed by the competency domain working groups are specific and measurable, such as prepare a research question and demonstrate knowledge of appropriate control, storage, and dispensing of investigational products. groups identified a total of 429 potential assessments across all domains, with 220 identified as appropriate for investigators and 209 identified as appropriate for crcs, a list that is in no way exhaustive. a considerable number of these assessments are identical for investigators and for crcs, so the total does not represent unique assessments. in terms of methods, many competency domain working groups suggested the use of case studies and observation of behavior, indicating that simply passing a quiz or multiple - choice exam is not indicative of mastery ; some also recommended pre / post testing. it is important to acknowledge that while the creation of detailed assessment methods was not within the scope of this work, some working groups did suggest methods that might be employed. competency domain working groups were also tasked with identifying and examining existing training believed to address the competencies identified within the groups respective competency domains. they took a broad approach to this task, based on the expertise and experiential knowledge of their members. it was not possible to examine every training program that might exist, but the groups reviewed a reasonably representative sample of offerings. in general, included were training and education offered by ctsa institutions ; by professional organizations devoted to education and resources ; by industry ; and by government units (see http://bit.ly/1iv821x, entitled training gaps by competency domains). collectively, competency domain working groups examined the following number of education and training offerings (with some offerings identified as appropriate for multiple competencies) (see table 2).table 2summary of existing education and trainingsctsa training and educationprofessional organizations education and trainingindustry education and traininggovernment education and training132140863total=343 offerings (not unique)ctsa, clinical and translational science awards. from table 2, of the 343, 219 unique education and training offerings were identified. every competency domain had associated training with a few exceptions, noted below: leadership, professionalism, and team science : identify and apply professional guidelines and codes of ethics as they relate to the conduct of clinical trials. no training identified. leadership, professionalism, and team science : describe the methods necessary to work effectively with multidisciplinary and interprofessional research teams. no training identified. communication : describe the component parts of a traditional scientific publication. leadership, professionalism, and team science : identify and apply professional guidelines and codes of ethics as they relate to the conduct of clinical trials. leadership, professionalism, and team science : describe the methods necessary to work effectively with multidisciplinary and interprofessional research teams. appear to be available online ; not all of those offerings are readily accessible in the public domain, or free of charge, however. in addition to the trainings identified as meeting - specified competencies, groups cited 214 supplemental resources, including web sites, reports, books, and published articles. see http://bit.ly/1u3pybs for the catalog of identified existing education and trainings organized by competency domain. sample online offerings:northwestern university, clinical and translational sciences institute introduction to clinical research online modulesuniversity of washington, institute of translational health sciences (iths), self - directed learning centeroffice of research integrity : the lab, the research clinic nih : teaching the responsible conduct of research acrp : gcp an introduction to ich gcp guidelines collaborative institutional training initiative (citi) : populations in research requiring additional consideration uc davis : strengthening provider patient communication skills in clinical trials. northwestern university, clinical and translational sciences institute introduction to clinical research online modules university of washington, institute of translational health sciences (iths), self - directed learning center office of research integrity : the lab, the research clinic nih : teaching the responsible conduct of research acrp : gcp an introduction to ich gcp guidelines collaborative institutional training initiative (citi) : populations in research requiring additional consideration uc davis : strengthening provider patient communication skills in clinical trials. competency domain working groups were not tasked to consider cost as a factor in the groups work, but it is certainly an important consideration and some groups did so. the cost of these offerings ranges from free to a significant financial investment by the department or individual. similarly, the working groups were not tasked to consider training quality, but many did so nevertheless identifying the need for training that actively engages the learner, going beyond mere rote memorization. a number of competency domain working groups noted that existing training focuses too heavily on theoretical concepts and historical events and not enough on the application of knowledge. competency domain working groups frequently noted that online offerings are insufficient alone and must be supplemented by local institutional education and training. the education and training offerings that are listed in this report (see http://bit.ly/1u3pybs) are not endorsed by the ecrptq leadership team. it was outside the parameters of this project to conduct a thorough evaluation of the suggested trainings from the competency domain working groups. there was broad agreement that the assessment of their quality should be undertaken as a future phase of the project. competency domain working groups identified 115 specific training gaps across the 8 reviewed domains. in the review of identified trainings, 5 broad categories of gaps were identified : (1) training that is needed but does not currently exist ; (2) existing training that is not adequate ; (3) certification, documentation of skill, or formal assessment is needed ; (4) a core training curriculum needs to be defined and/or developed ; and (5) there is inadequate training at a level for crcs. overwhelmingly, groups concluded that some training exists for most ecrptq competency domains, but that this training is not adequate to fully meet the needs of the investigators and crcs. this finding is consistent with the data collected in the jtf core competency survey ; participants were asked whether they felt a need for training in these domains, with the majority of respondents indicating they felt training was needed. overall, the education and training identified is primarily investigator focused, particularly offerings provided by ctsa institutions. lack of training is keenly felt in the adoption of new technologies and in areas such as data management. training is not generally organized by level of expertise, and often does not distinguish roles and responsibilities between investigators and crcs. some competency domain working groups, such as leadership, professionalism, and team science, and communication were forced to draw from offerings completely outside the context of clinical research, because they were unable to find relevant training within clinical research. as noted previously, many of the trainings cited in this report do not provide opportunities to apply knowledge and do not incorporate learning strategies known to be effective in promoting the development of competence. the s / br working group reviewed all the competency domain working groups deliverables and offered suggested edits to the ecrptq competencies in some domains to promote greater inclusion of research teams carrying out clinical trials involving behavioral interventions and assessments. additional comments made by the group regarding trainings and assessments for select domains were made (reference to http://bit.ly/1raesik). the recently completed jtf core competency survey that is currently being analyzed will provide important data that will help validate the jtf framework. the survey asked individuals to self - assess their own level of competence in the framework s domains, as well as the relevance of those domains and their perceived needs for additional training. this organization is also currently exploring mechanisms to revise and update its competency statements, particularly as new scientific fields and technologies emerge.building upon the identification of assessment areas by competency domain working groups, specific assessments must be developed to assess competence. such assessments should focus not only on different study team member roles, but levels of mastery as well.an evaluation of the quality of existing training should be undertaken, as well as an expansion of the catalog of training that emerged from the working groups. this catalog does not include all available educational opportunities and should be expanded to include additional relevant training.a deeper exploration of training gaps should be undertaken to determine whether new training modules are needed. if so, the development of this education and training should be undertaken by individuals skilled in instructional design and curriculum development, built upon the principles of adult learning.examination of a cloud - based learning management platform to support individuals seeking and tracking their cbe and assessment, based on the competency framework developed in this work may be warranted.an eportfolio system would be identified to allow pis and crcs to collect, organize, and share their completed trainings, demonstrate learning, and have a portable record of their achievements. such a system would allow individuals to upload artifacts of competence that could be made available to relevant institutions, sponsors, and other regulatory bodies.because of the extraordinary opportunity of working with external stakeholder organizations to identify standard competencies for these 2 cohorts (investigators and crcs), these continued activities should be undertaken in partnership with individuals from the jtf, coapcr, acrp, clinical trials transformation initiative (ctti) and other organizations invested in clinical research professional training. these organizations are committed to study team education and training, and the jtf framework is gaining significant traction nationally and internationally through their efforts.recommendations for institutional policies on clinical research training should be expanded beyond basic irb and gcp training, with step - wise approaches to training personnel.job descriptions should be modified to reflect specific competencies by levels.inter-institutional training courses should be developed and shared as cost - free, easily accessible, web - based formats with additional train - the - trainer mechanisms for onsite training and continuing education.academic pathways for baccalaureate and graduate degrees in clinical research should be endorsed and more highly accessible to clinical research professionals working in academic medical centers and hospitals. if an institution does not offer a clinical research program of study, then clinical research staff should be able to transfer educational benefits to other institutions. the recently completed jtf core competency survey that is currently being analyzed will provide important data that will help validate the jtf framework. the survey asked individuals to self - assess their own level of competence in the framework s domains, as well as the relevance of those domains and their perceived needs for additional training. this organization is also currently exploring mechanisms to revise and update its competency statements, particularly as new scientific fields and technologies emerge. building upon the identification of assessment areas by competency domain working groups such assessments should focus not only on different study team member roles, but levels of mastery as well. an evaluation of the quality of existing training should be undertaken, as well as an expansion of the catalog of training that emerged from the working groups. this catalog does not include all available educational opportunities and should be expanded to include additional relevant training. a deeper exploration of training gaps should be undertaken to determine whether new training modules are needed. if so, the development of this education and training should be undertaken by individuals skilled in instructional design and curriculum development, built upon the principles of adult learning. examination of a cloud - based learning management platform to support individuals seeking and tracking their cbe and assessment, based on the competency framework developed in this work may be warranted. an eportfolio system would be identified to allow pis and crcs to collect, organize, and share their completed trainings, demonstrate learning, and have a portable record of their achievements. such a system would allow individuals to upload artifacts of competence that could be made available to relevant institutions, sponsors, and other regulatory bodies. because of the extraordinary opportunity of working with external stakeholder organizations to identify standard competencies for these 2 cohorts (investigators and crcs), these continued activities should be undertaken in partnership with individuals from the jtf, coapcr, acrp, clinical trials transformation initiative (ctti) and other organizations invested in clinical research professional training. these organizations are committed to study team education and training, and the jtf framework is gaining significant traction nationally and internationally through their efforts. recommendations for institutional policies on clinical research training should be expanded beyond basic irb and gcp training, with step - wise approaches to training personnel. inter - institutional training courses should be developed and shared as cost - free, easily accessible, web - based formats with additional train - the - trainer mechanisms for onsite training and continuing education. academic pathways for baccalaureate and graduate degrees in clinical research should be endorsed and more highly accessible to clinical research professionals working in academic medical centers and hospitals. if an institution does not offer a clinical research program of study, then clinical research staff should be able to transfer educational benefits to other institutions. competency domain working groups identified 115 specific training gaps across the 8 reviewed domains. in the review of identified trainings, 5 broad categories of gaps were identified : (1) training that is needed but does not currently exist ; (2) existing training that is not adequate ; (3) certification, documentation of skill, or formal assessment is needed ; (4) a core training curriculum needs to be defined and/or developed ; and (5) there is inadequate training at a level for crcs. overwhelmingly, groups concluded that some training exists for most ecrptq competency domains, but that this training is not adequate to fully meet the needs of the investigators and crcs. this finding is consistent with the data collected in the jtf core competency survey ; participants were asked whether they felt a need for training in these domains, with the majority of respondents indicating they felt training was needed. overall, the education and training identified is primarily investigator focused, particularly offerings provided by ctsa institutions. lack of training is keenly felt in the adoption of new technologies and in areas such as data management. training is not generally organized by level of expertise, and often does not distinguish roles and responsibilities between investigators and crcs. some competency domain working groups, such as leadership, professionalism, and team science, and communication were forced to draw from offerings completely outside the context of clinical research, because they were unable to find relevant training within clinical research. as noted previously, many of the trainings cited in this report do not provide opportunities to apply knowledge and do not incorporate learning strategies known to be effective in promoting the development of competence. the s / br working group reviewed all the competency domain working groups deliverables and offered suggested edits to the ecrptq competencies in some domains to promote greater inclusion of research teams carrying out clinical trials involving behavioral interventions and assessments. additional comments made by the group regarding trainings and assessments for select domains were made (reference to http://bit.ly/1raesik). the recently completed jtf core competency survey that is currently being analyzed will provide important data that will help validate the jtf framework. the survey asked individuals to self - assess their own level of competence in the framework s domains, as well as the relevance of those domains and their perceived needs for additional training. this organization is also currently exploring mechanisms to revise and update its competency statements, particularly as new scientific fields and technologies emerge.building upon the identification of assessment areas by competency domain working groups, specific assessments must be developed to assess competence. such assessments should focus not only on different study team member roles, but levels of mastery as well.an evaluation of the quality of existing training should be undertaken, as well as an expansion of the catalog of training that emerged from the working groups. this catalog does not include all available educational opportunities and should be expanded to include additional relevant training.a deeper exploration of training gaps should be undertaken to determine whether new training modules are needed. if so, the development of this education and training should be undertaken by individuals skilled in instructional design and curriculum development, built upon the principles of adult learning.examination of a cloud - based learning management platform to support individuals seeking and tracking their cbe and assessment, based on the competency framework developed in this work may be warranted.an eportfolio system would be identified to allow pis and crcs to collect, organize, and share their completed trainings, demonstrate learning, and have a portable record of their achievements. such a system would allow individuals to upload artifacts of competence that could be made available to relevant institutions, sponsors, and other regulatory bodies.because of the extraordinary opportunity of working with external stakeholder organizations to identify standard competencies for these 2 cohorts (investigators and crcs), these continued activities should be undertaken in partnership with individuals from the jtf, coapcr, acrp, clinical trials transformation initiative (ctti) and other organizations invested in clinical research professional training. these organizations are committed to study team education and training, and the jtf framework is gaining significant traction nationally and internationally through their efforts.recommendations for institutional policies on clinical research training should be expanded beyond basic irb and gcp training, with step - wise approaches to training personnel.job descriptions should be modified to reflect specific competencies by levels.inter-institutional training courses should be developed and shared as cost - free, easily accessible, web - based formats with additional train - the - trainer mechanisms for onsite training and continuing education.academic pathways for baccalaureate and graduate degrees in clinical research should be endorsed and more highly accessible to clinical research professionals working in academic medical centers and hospitals. if an institution does not offer a clinical research program of study, then clinical research staff should be able to transfer educational benefits to other institutions. the recently completed jtf core competency survey that is currently being analyzed will provide important data that will help validate the jtf framework. the survey asked individuals to self - assess their own level of competence in the framework s domains, as well as the relevance of those domains and their perceived needs for additional training. this organization is also currently exploring mechanisms to revise and update its competency statements, particularly as new scientific fields and technologies emerge. building upon the identification of assessment areas by competency domain working groups such assessments should focus not only on different study team member roles, but levels of mastery as well. an evaluation of the quality of existing training should be undertaken, as well as an expansion of the catalog of training that emerged from the working groups. this catalog does not include all available educational opportunities and should be expanded to include additional relevant training. a deeper exploration of training gaps should be undertaken to determine whether new training modules are needed. if so, the development of this education and training should be undertaken by individuals skilled in instructional design and curriculum development, built upon the principles of adult learning. examination of a cloud - based learning management platform to support individuals seeking and tracking their cbe and assessment, based on the competency framework developed in this work may be warranted. an eportfolio system would be identified to allow pis and crcs to collect, organize, and share their completed trainings, demonstrate learning, and have a portable record of their achievements. such a system would allow individuals to upload artifacts of competence that could be made available to relevant institutions, sponsors, and other regulatory bodies. because of the extraordinary opportunity of working with external stakeholder organizations to identify standard competencies for these 2 cohorts (investigators and crcs), these continued activities should be undertaken in partnership with individuals from the jtf, coapcr, acrp, clinical trials transformation initiative (ctti) and other organizations invested in clinical research professional training. these organizations are committed to study team education and training, and the jtf framework is gaining significant traction nationally and internationally through their efforts. recommendations for institutional policies on clinical research training should be expanded beyond basic irb and gcp training, with step - wise approaches to training personnel. inter - institutional training courses should be developed and shared as cost - free, easily accessible, web - based formats with additional train - the - trainer mechanisms for onsite training and continuing education. academic pathways for baccalaureate and graduate degrees in clinical research should be endorsed and more highly accessible to clinical research professionals working in academic medical centers and hospitals. if an institution does not offer a clinical research program of study, then clinical research staff should be able to transfer educational benefits to other institutions. this document is the result of the ctsa consortium ecrptq supplement and includes discussion and consensus documents with iterative revisions. the competencies and assessments generated in phase ii of the ecrptq supplement have been approved by the ctsa consortium and have the support of jtf, coapcr, and acrp. the competencies and assessments have been submitted to national center for advancing translational sciences (ncats) for their consideration. although these documents provide a framework for investigator and crc training it does not provide direction on how to implement a training program. the purpose of this document is to provide a standard set of core clinical trial competencies and ksa assessments that equip investigators and crcs with the necessary skills to more effectively, efficiently, and safely execute clinical trials. education and training should be patterned to these competencies and ultimately lead to specific formative and summative evaluations of learning. | introductiontraining for the clinical research workforce does not sufficiently prepare workers for today s scientific complexity ; deficiencies may be ameliorated with training. the enhancing clinical research professionals training and qualifications developed competency standards for principal investigators and clinical research coordinators.methodsclinical and translational science awards representatives refined competency statements. working groups developed assessments, identified training, and highlighted gaps.resultsforty-eight competency statements in 8 domains were developed.conclusionstraining is primarily investigator focused with few programs for clinical research coordinators. lack of training is felt in new technologies and data management. there are no standardized assessments of competence. |
polybrominated diphenyl ethers (pbdes) are additive flame retardant chemicals used since the 1970s in commercial and household products. the technical formulation pentabde is composed of pbde congeners containing three to six bromines, primarily bde-28, bde-47, bde-99, bde-100, and bde-153. it was used in furniture containing polyurethane foam to meet fire standards such as california technical bulletin 117. bde-153 also occurs in the octabde technical formulation used in electronics. of the worldwide production, 95% of the pentabde produced was consumed in north america, where concentrations of several pentabde congeners in the environment and people are approximately an order of magnitude higher than those reported in europe or asia. because of their persistence, lipophilicity, ability to bioaccumulate, and concerns regarding adverse effects on human health, production of pentabde and octabde is now prohibited by the stockholm convention, an international treaty that governs persistent organic pollutants. chemical manufacturers withdrew pentabde and octabde from production in 2004. despite the current restrictions, human exposure is still occurring due to the release of these flame retardant chemicals from existing products and through contaminated foods. in the united states, unlike the highly brominated bde-183 and bde-209 that have half - lives on the order of weeks to months, the half - lives of the major pentabde congeners have not been directly measured in humans, but have been estimated to be on the order of years. these half - life estimates are uncertain because the calculations assumed steady state conditions and compared body burdens with uncertain exposure estimates. toxicological studies have demonstrated that pbdes, particularly of the pentabde formulations, adversely affect endocrine homeostasis and neurodevelopment, and have reproductive effects. recently, epidemiological studies conducted in the united states have linked pbde exposure to adverse effects on neurodevelopment, and altered thyroid and reproductive hormone levels. exposure measurement error occurs when a study participant is assigned an exposure that is different from their true exposure over the biologically relevant time period. such error, even if independent of outcome, can lead to biased effect estimates. it may even completely remove a true association between an exposure and outcome of interest. with continuous exposures, epidemiologists typically use either a continuous exposure measure (e.g., serum concentration) or place participants into categories (e.g., low, medium, or high). kappa statistics quantify the amount of agreement between an initial exposure categorization and an exposure categorization at a later point in time (e.g., did a participant in the high exposure category remain in the high exposure category later). if the amount of variability over time within subjects is small compared to variability between subjects, the icc will be close to 1 and the exposure metric is considered reliable. for example, a high icc indicates that highly exposed people tend to remain high relative to other people. there are currently no studies of this kind that have evaluated the potential amount of pbde exposure misclassification in epidemiological studies. our objectives were to characterize pbde serum concentrations in a new england cohort and assess temporal variability of this exposure biomarker over a one - year period. additionally, we assessed demographic characteristics and serum lipid concentrations as predictors of serum pbde concentrations. we recruited a convenience sample of 52 adults living and working in the greater boston (ma, united states) metropolitan area from winter 2010 to summer 2011 to participate in the flame retardant exposure study (flare study). eligible subjects had to be healthy, nonsmoking adults over the age of 18, working in an office environment at least 20 h a week, and planning to reside in the greater boston metropolitan area for the study duration. participants were excluded for having a prior diagnosis of thyroid or male reproductive disease or if they were pregnant. the city of boston requires that furniture in public spaces meet certain fire codes. we conducted three sampling rounds : round 1 (1/13/104/15/10), round 2 (6/3/109/15/10), and round 3 (1/31/114/27/11). serum samples were provided by 49 of 51 (96%) participants in round 1, 50 of 52 (96%) participants in round 2, and 42 of 52 (81%) participants in round 3. the missing blood samples were due to the following reasons : phlebotomist was unable to conduct venipuncture, participant declined, participant moved out of study area, or participant could no longer be contacted. all blood samples were nonfasting. during each sampling visit, study personnel administered a questionnaire designed to collect basic demographic and health information. the boston university medical center institutional review board approved the study protocol and all subjects gave written informed consent prior to participation. the involvement of the centers for disease control and prevention (cdc) laboratory was determined not to constitute engagement in human subjects research. a trained phlebotomist collected 30 ml of blood from each participant during each sampling round. blood samples were processed on the day of collection and serum samples were stored at 80 c in amber glass vials until analysis. to eliminate potential issues with interassay variability, serum samples collected from all three rounds were analyzed at one time following round 3. serum samples were analyzed for lipids (total triglycerides, total cholesterol) and 11 pbde congeners (bde-17, bde-28, bde-47, bde-66, bde-85, bde-99, bde-100, bde-153, bde-154, bde-183, bde-209) at the cdc using established methods. final analytic determination of the pbde congeners was performed by gas chromatography isotope dilution high - resolution mass spectrometry using a mat95xp (thermofinnigan mat, bremen, germany) instrument. samples were randomized and analyzed with quality control (qc) (n = 3) and blank samples (n = 3) in each batch of 24 unknowns. all concentration data were reported as background subtracted, where correction was made based on the median amount present in blank samples. limits of detection (lod) were calculated as the highest of two methods : (i) 3 times the standard deviation of the method blank samples and (ii) as the lowest point in the calibration curve having a signal - to - noise ratio greater than 3 (primarily for analytes with low to no detectable method blank concentration). for measurements below the lod, we substituted 1/2 lod. pbde congeners were log - normally distributed, as identified by histograms and shapiro wilks tests, and thus log - transformed. we calculated round - specific geometric means (gm) and geometric standard deviations (gsd) for congeners detected in > 50% of the samples. pbdes is defined here as the sum of bde-28, bde-47, bde-99, bde-100, and bde-153. all statistical analyses were conducted using sas version 9.3 (sas institute, cary, nc, usa). we calculated kappa statistics to assess the amount of agreement between exposure categories in round 1 and round 3, the two winter time points. we used three exposure classification schemes : median (low, high), tertile (low, medium, high) and quartile (low, medium, high, very high). kappa statistics range from 0 to 1, with 1 indicating perfect agreement between two observations. these analyses were restricted to participants who provided a blood sample in both sampling rounds (participants = 40, serum samples = 80). contingency tables were constructed to display the level of agreement between each subject s initial and final exposure category. we report the weighted kappa, instead of the simple kappa, when assessing agreement for tertiles and quartiles. we used the following general linear model with a random intercept to estimate the between- and within - subject variance components associated with pbde congener levels over the study period:1where yij represents the natural logarithm of the pbde congener level of the ith participant on the jth round of sampling, 0 is the fixed effect intercept, bi is the random intercept of the ith individual, and ij is the random error. to determine how serum lipids affect the variance components, we added a predictor variable, lipidij, the lipid level of the ith participant on the jth sampling round:2we estimated the intraclass correlation coefficient (icc) to assess reliability of serum pbde congener concentrations using the following formula:3where b is the between - subject variance and w is the within - subject variance. we also determined whether (i) average biomarker levels increased or decreased by study round or (ii) if congener concentrations were associated with predictor variables obtained from questionnaires. rather than standardizing pbde measurements to lipids, we adjusted for lipid as a covariate in regression models, allowing us to estimate the effect of this variable. to evaluate the fixed effects of time and covariates, we used the following model:4where yij, 0, bi, and ij are defined as earlier. time2 and time3 are indicator variables : 1 is the average difference of the log(pbde) measurement from round 1 to round 2 ; 2 is the average difference of the log(pbde) measurement from round 1 to round 3. age is the age of the ith participant at the initial sampling round (categorized as 37 or 50% : bde-28, bde-47, bde-99, bde-100, and bde-153. bde-47 had the highest serum concentration followed by bde-153 ; both were detected in 100% of serum samples. bde-99, bde-100, and bde-28 had detection frequencies of 92%, 89%, and 68%, respectively. detection rates for bde-17, bde-66, bde-85, bde-154, bde-183, and bde-209 ranged from 1% to 22% and were not further analyzed. supporting information (si) table 1 presents detection rates, lods, and ranges for all analyzed congeners. additional congeners that were analyzed but detected infrequently (bde-17, bde-66, bde-85, bde-154, bde-183, bde-209) are presented in supporting information (si) table 1. means and standard deviation presented. table 3 presents the estimated iccs for the serum pbde congeners calculated using eqs 1 (unadjusted), 2 (adjusted for lipid), or 4 (adjusted for lipid and other covariates). the icc estimates were very high, particularly for pbdes, bde-47, and bde-153, ranging from 0.96 to 0.99. as a sensitivity analysis for missing data, we calculated iccs using eq 1 for the subset of individuals that contributed three serum measurements (n = 40) and the results were similar (not shown). see si figure 1 for a graph of individual data for bde-47 and si figure 2 for a simple correlation analysis for this congener. participants = 52, serum samples = 142. equation (1) : yij = 0 + bi + ij. equation (2) : yij = 0 + 1lipid + bi + ij. equation (3) : yij = 0 + 1time2 + 2time3 + 3agei + 4sexi + 5bmii + 6lipidij + bi + ij. table 3 also presents the kappa statistics quantifying the agreement between exposure categorization in round 1 and round 3 (approximately one year apart). kappa statistics between 0.61 and 0.80 are considered in substantial agreement and kappa statistics > 0.80 are considered in almost perfect agreement. for example, the kappa statistics for bde-47, based on exposure categorization by median (e.g., low, high), tertile (e.g., low, medium, high), or quartile (e.g., low, medium, high, very high), were 0.80, 0.67, and 0.84, respectively. si figure 3 presents the contingency tables associated with the kappa statistics for bde-47. we also calculated kappa statistics for each congener comparing round 1 and round 2 (6 months apart) and the results were similar (si table 3). table 4 presents parameter estimates and p - values for regression models (eq 4) predicting pbde levels as a function of sampling round, serum lipids, age, sex, and bmi. exponentiating the beta coefficients, we find that bde-47 and bde-100 levels significantly decreased by 7.9% (p = 0.029) and 13.9% (p = 0.024), respectively, from round 2 to round 3. while there were no other significant temporal changes, all congeners except bde-99 had negative slopes from round 2 to round 3 and positive trends from round 1 to round 2. we also ran regression analysis using pbdes standardized to lipids and our results were similar to the presented model with lipid as a covariate (not shown). using akaike information criterion (aic) as a guide, we found that evaluating time as a categorical variable (instead of continuous in months, e.g. j = month) provided the better fit, though results were similar in both models (not shown). we also analyzed these data using a model with continuous time adding a covariate for season (summer vs winter), and did not find any significant trends (not shown). results were similar when analyzed using only individuals that contributed three serum measurements (not shown). abbreviations : (pg / ml) picogram per milliliter serum ; (ref) reference group ; (se) standard error ; (yo) years old. exp() yields the multiplicative increase in pbde per unit change in predictor, e.g., exp(0.082) = 0.921 = 7.9% decrease. for example, bde-47 increased 0.15% (p 80%), and study dropout was classified as mcar, this limitation was unlikely to have biased our results. our study sample size (52 participants, 142 serum samples) limited the amount of variables we could evaluate simultaneously in regression models and likely reduced our power to detect statistically significant associations. participants self - reported height and weight at the beginning of the study, potentially introducing error into the estimate of bmi. with only one estimate of bmi, we can not examine changes in weight (or adipose tissue) as a determinant of pbde serum concentrations we used a convenience sample of office workers in the boston area that were mostly white, not overweight, and highly educated, and we can not be certain our results can be generalized to the u.s. our study demonstrates that a single pbde serum measurement of bde-28, bde-47, bde-99, bde-100, and bde-153 reliably estimates a participant s blood concentration over a one - year period. we also observed decreases of serum concentrations of some pbde congeners that were not explained by changes in serum lipid. | polybrominated diphenyl ethers (pbdes) are flame retardant chemicals used in consumer products. they are common contaminants in human serum and associated with adverse health effects. our objectives were to characterize pbde serum concentrations in a new england cohort and assess temporal variability of this exposure biomarker over a one - year period. we collected three repeated measurements at six - month intervals from 52 office workers from the greater boston (ma, united states) area from 2010 to 2011. the intraclass correlation coefficient for bdes 28, 47, 99, 100, and 153 ranged from 0.87 to 0.99, indicating that a single serum measurement can reliably estimate exposure over a one - year period. this was true for both lipid adjusted and nonlipid adjusted concentrations. the kappa statistics, quantifying the level of agreement of categorical exposure classification, based on medians, tertiles, or quartiles ranged from 0.67 to 0.90. some congeners showed nonsignificant increases from sampling round 1 (winter) to round 2 (summer) and significant decreases from round 2 to round 3 (winter). this study highlights the high reliability of a single serum pbde measurement for use in human epidemiologic studies. |
a link between venous thromboembolism (vte) and anti - neutrophil cytoplasmic antibody (anca)-associated vasculitis (aav) is well established. this showed an incidence of vte of 7 per 100 patient years, which is significantly higher than that described for normal population cohorts or for patients with rheumatoid arthritis or systemic lupus erythematosus. in addition, weidner. reported that 13 of 105 patients with active aav had thromboembolic events during the period of active disease. more recently, a retrospective study confirmed an increased risk of developing vtes at 1.8 per 100 patient years overall, increasing to 6.7 during active disease, which also appeared independent of classic risk factors. none of these patients had renal vein thrombosis and we are not aware of any previous reports of this. a novel contributing mechanism to vte has been demonstrated in aav patients, which together with the increased endothelial activation could increase the risk of vte. antibodies against plasminogen antibodies and tissue plasminogen activator antibodies were found in the plasma of aav patients and caused functional inhibition of fibrinolysis in vitro. a 24-year - old afro - caribbean man presented with a 4-week history of fever, night sweats and abdominal pain associated with some weight loss and loose stools. on admission, his serum creatinine was 220 mol / l with a urine protein creatinine ratio (pcr) of 306 mg / mmol. g / l and c - reactive protein (crp) was raised at 121 mg / l. his urinalysis revealed haematuria (4 +) and he had an atypical anca but was negative for antibodies to both myeloperoxidase (mpo) and proteinase-3 (pr3). serum protein electrophoresis showed a reduced albumin but no increase in 2-macroglobulin or other abnormality. a renal ultrasound showed normal - sized kidneys, and in view of his fever and abdominal pain, he underwent a computed tomography scan. unexpectedly, this revealed bilateral pulmonary emboli and bilateral renal vein thrombosis as shown in figure 1. his initial thrombophilia screen showed a lupus anticoagulant (la) by dilute - activated partial thromboplastin time analysis with negative anti - cardiolipin antibodies (acl). the acute kidney injury was felt to be due to the renal vein thrombosis for which he was anticoagulated, initially with heparin and then with warfarin, and discharged. a month after his initial presentation, he was re - admitted with vomiting, possibly secondary to warfarin intolerance, and a subtherapeutic international normalised ratio requiring intravenous unfractionated heparin. his renal function was unchanged with a serum creatinine of 180 mol / l, crp was now < 5 mg / l, urine pcr was 363 mg / mmol and serum albumin 32 g / l. tubular atrophy was in 10% of the sample and immunoperoxidase staining was negative for all immunoglobulin and complement components. the possibility of immunosuppression was considered. however, at this point, his serum creatinine fell progressively and, 2 months after the renal biopsy, was in the normal range at 102 mol / l, where it stayed for the next 5 months. in view of this, and the fact that the renal biopsy had shown largely inactive lesions, he was not given immunosuppression. the possibility of another renal biopsy was being considered to assess if there were any active lesions but he then failed to attend clinic and, despite considerable efforts to contact him, was lost to follow - up. he re - presented 28 months after initial presentation with irreversible kidney damage, which was confirmed on another renal biopsy. he has remained anca positive, and his anti - mpo and pr3 levels were repeated with an alternative assay. although these had previously been negative using the phadia elia assay, he was mpo positive with the fidis luminex assay. his la has been confirmed and, although acl were negative at initial presentation, they were later found to be positive on more than one occasion. he has had no further thrombotic events during follow - up though has remained on anticoagulants. this is the unusual case of aav presenting with pulmonary emboli and bilateral renal vein thrombosis. although not appreciated on the initial tests, he was in fact positive for antibodies to mpo on further testing. an increased incidence of la and acl has been described in systemic vasculitis with 25 of 144 patients positive for one of these tests at one time and 9 of these having clinical apls. it is not clear if aav itself predisposes to the development of the apls or if aav develops as a result of apls. however, a very small proportion of apls patients have aav, so, the former seems more likely. in this patient, the procoagulant state due to the presence of anti - phospholipid antibodies and aav combined to cause renal vein thrombosis and pulmonary emboli. it should be noted that renal vein thrombosis is most commonly seen with either malignancy or the nephrotic syndrome. in one single - centre series of 218 patients with renal vein thrombosis, 143 had malignancy and 43 had nephrotic syndrome. only 36 patients in this study underwent thrombophilia screening, making conclusions on a link between renal vein thrombosis and la or acl difficult. we do not know the degree of his proteinuria prior to his renal vein thrombosis but nephrotic range proteinuria is unusual with renal aav and would be unlikely given the later findings on renal biopsy. g / l but this was in the context of an inflammatory response, which would have lowered his albumin further. nonetheless, it is possible that his proteinuria represented an additional risk factor for thrombosis. this case illustrates the known link between aav and vte and emphasizes that other risk factors for venous thrombosis such as anti - phospholipid antibodies may add to the procoagulant state in aav. the coexistence of an la and acl led to renal vein thrombosis, which is otherwise predominantly seen in patients with malignancy or the nephrotic syndrome. in addition, a positive anca with negative anti - mpo or pr3 assays may still be significant, as our patient was positive for anti - mpo antibodies when tested with a second assay. finally, the progression to end - stage kidney disease emphasizes the importance of close monitoring for all patients with aav. | we report a case of anti - neutrophil cytoplasmic antibody (anca)-associated necrotizing crescentic glomerulonephritis presenting with bilateral renal vein thrombosis and pulmonary emboli in a patient who also had a lupus anticoagulant and anti - cardiolipin antibodies. although the link between venous thrombosis and anca vasculitis is well established, the coexistence of renal vein thrombosis is unusual. furthermore, despite the positive anca, he was initially negative for antibodies to myeloperoxidase (mpo) and proteinase-3 (pr3), illustrating that a positive anca may be significant despite a negative test for antibodies to mpo and pr3. |
thirty - five patients with poag treated with pg analogues and 35 age - matched healthy control subjects were included in this cross - sectional study undertaken at a single university hospital between november 2011 and july 2012. informed consent was obtained from all patients, and the study was carried out with the approval from the institutional review board. participants were divided into two groups : group 1 was composed of patients, who had been exclusively treated with pg analogues for at least 2 years without any prior use of another class of glaucoma medication ; group 2 was composed of healthy age - matched controls without any systemic or ocular disease. all patients were underwent a complete ophthalmic examination including slit - lamp examination, iop determination and fundus examination. poag was defined as open - angle detected by gonioscopy, an iop > 21 mmhg with goldmann applanation tonometer and characteristic glaucomatous optic nerve head changes (rim thinning, excavation, and/or retinal nerve fiber layer defects) and/or visual field defects were detected on 2 consecutive tests. subjects who used any iop - lowering drugs except pg analogues, those with any corneal pathology on slit - lamp examination, those with a history of ocular surgery or ocular trauma, those who were using contact lenses were excluded from the study. control subjects were recruited during routine screening visits and had no history of ocular disease and pathologic ocular findings apart from cataract, normal appearing optic discs, normal visual fields and iop measurements below 21 mmhg. in vivo confocal microscopy of the cornea was performed using confoscan 3.0 (vigonza, italy) attached to an immersion lens (achroplan 40x/0.75 w, zeiss, oberkochen, germany). the immersion lens had a working distance of 1.98 mm, a numerical aperture of 0.75, and the front surface area of 16.61 mm. data were collected for both eyes, and one eye for each patient was chosen at random. before examination, each cornea was anesthetized with propacaine hydrochloride (alcaine 0.5%, alcon laboratories inc., belgium). after application of viscotears (carbomer 0.2%, novartis, basel, switzerland) as a coupling agent to the applanating lens, for each subject, four to six complete full - layer corneal scans were obtained. the images represented an area of 450 m 340 m, had a lateral resolution of 1 m, and a depth resolution of 10 m. the mean magnification obtained was 500x on a 15 inch display (1024 768 pixels). the best - focused two images for each stromal layer were selected for analysis, and the mean value was calculated. keratocyte density was determined using a manual method of counting images of cell nuclei in a predefined area of confocal images, using the software provided with confoscan 3.0. cells that overlapped the boundary box were counted at only the superior and the left half of the box. keratocytes densities were measured at the anterior (0100 m posterior to the basal epithelium), middle (half the distance between the basal epithelium and the endothelium), and posterior (0100 m anterior to the endothelium) stromal layers. central corneal thickness was measured in all subjects with an ultrasonic pachymeter (nidek echoscan us-4000). to avoid the effects of the diurnal variation, all measurements were carried out at the same time of day (between 9:00 am and 12:00 am). in line with previously published methodology, cct measurements were taken using ultrasound pachymetry by a single observer experienced in this field. for each patient, after instillation of a topical anesthetic (alcaine 0.5%, alcon laboratories inc., data analysis was performed by spss 15.0 (statistical package for social sciences, spss inc. the data were statistically analyzed using student 's t - test and chi - square test. correlations between keratocytes density and cct were analyzed using pearson 's correlation coefficients and a p = 0.05 was accepted as statistically significant. thirty - five patients with poag treated with pg analogues and 35 age - matched healthy control subjects were included in this cross - sectional study undertaken at a single university hospital between november 2011 and july 2012. informed consent was obtained from all patients, and the study was carried out with the approval from the institutional review board. participants were divided into two groups : group 1 was composed of patients, who had been exclusively treated with pg analogues for at least 2 years without any prior use of another class of glaucoma medication ; group 2 was composed of healthy age - matched controls without any systemic or ocular disease. all patients were underwent a complete ophthalmic examination including slit - lamp examination, iop determination and fundus examination. poag was defined as open - angle detected by gonioscopy, an iop > 21 mmhg with goldmann applanation tonometer and characteristic glaucomatous optic nerve head changes (rim thinning, excavation, and/or retinal nerve fiber layer defects) and/or visual field defects were detected on 2 consecutive tests. subjects who used any iop - lowering drugs except pg analogues, those with any corneal pathology on slit - lamp examination, those with a history of ocular surgery or ocular trauma, those who were using contact lenses were excluded from the study. control subjects were recruited during routine screening visits and had no history of ocular disease and pathologic ocular findings apart from cataract, normal appearing optic discs, normal visual fields and iop measurements below 21 mmhg. in vivo confocal microscopy of the cornea was performed using confoscan 3.0 (vigonza, italy) attached to an immersion lens (achroplan 40x/0.75 w, zeiss, oberkochen, germany). the immersion lens had a working distance of 1.98 mm, a numerical aperture of 0.75, and the front surface area of 16.61 mm. data were collected for both eyes, and one eye for each patient was chosen at random. before examination, each cornea was anesthetized with propacaine hydrochloride (alcaine 0.5%, alcon laboratories inc., belgium). after application of viscotears (carbomer 0.2%, novartis, basel, switzerland) as a coupling agent to the applanating lens, the full thickness of the central cornea was scanned, by the same operator. for each subject, four to six complete full - layer corneal scans were obtained. the images represented an area of 450 m 340 m, had a lateral resolution of 1 m, and a depth resolution of 10 m. the mean magnification obtained was 500x on a 15 inch display (1024 768 pixels). the best - focused two images for each stromal layer were selected for analysis, and the mean value was calculated. keratocyte density was determined using a manual method of counting images of cell nuclei in a predefined area of confocal images, using the software provided with confoscan 3.0. cells that overlapped the boundary box were counted at only the superior and the left half of the box. keratocytes densities were measured at the anterior (0100 m posterior to the basal epithelium), middle (half the distance between the basal epithelium and the endothelium), and posterior (0100 m anterior to the endothelium) stromal layers. central corneal thickness was measured in all subjects with an ultrasonic pachymeter (nidek echoscan us-4000). to avoid the effects of the diurnal variation, all measurements were carried out at the same time of day (between 9:00 am and 12:00 am). in line with previously published methodology, cct measurements for each patient, after instillation of a topical anesthetic (alcaine 0.5%, alcon laboratories inc., data analysis was performed by spss 15.0 (statistical package for social sciences, spss inc. the data were statistically analyzed using student 's t - test and chi - square test. correlations between keratocytes density and cct were analyzed using pearson 's correlation coefficients and a p = 0.05 was accepted as statistically significant. thirty - five patients with poag (22 female, 13 male) and 35 control subjects (14 female, 21 male) were enrolled in this study. the mean duration of the pg analogue treatment was 6.3 3.4 years (range = 212 years) in the glaucoma group. the mean age of patients with glaucoma and control subjects were 65.2 7.1 years (range = 5077 years) and 64.4 4.6 years (range = 5982 years), respectively. there were no significant differences across the two groups with respect to age (p = 0.596) and gender (p = 0.94). the mean keratocyte densities in three stromal layers and cct values were significantly lower in patients with pg analogue therapy compared with control subjects [table 1, figs. 1 and 2 ]. twenty - six patients were being treated with latanoprost and 9 with travoprost eye drops. the mean age of patients as well as the duration of topical anti - glaucoma therapy, were similar between groups [table 2 ]. there were no significant differences observed in keratocyte densities and the cct measurements between who were on latanoprost versus travoprost medication [table 2 ]. a weak positive correlation between the keratocyte densities in each stromal layers and cct was observed in patients with pg analogue therapy (anterior stroma, = 0.412, p < 0.001 ; midstroma, = 0.572, p < 0.001 ; posterior stroma, = 0.547, p < 0.001). a negative but statistically insignificant correlation was found between the anterior stromal (= 0.014, p = 0.934), mid - stromal (= 0.319, p = 0.062) and posterior stromal (= 0.232, p = 0.179) keratocyte densities and duration of the pg analogue treatment. in vivo confocal microscopic images of anterior stromal keratocytes in a control (a) subject and patient treated with prostaglandin analogue (b) mid - stromal section from a healthy subject (a) and a patient on prostaglandin analogue treatment (b) comparison of the keratocyte densities and central corneal thickness measurements between glaucoma patients on pg analogue therapy and control subjects comparison of the keratocyte densities and central corneal thickness measurements between glaucoma patients on latanoprost versus travoprost therapy in this study, keratocyte densities and cct were found to be lower in poag patients on pg analogue therapy for at least 2 years compared with normal subjects. overall, our results suggest a possible deleterious effect of topical pg analogues on keratocytes in the corneas of poag patients. our study is noteworthy in that the study subjects had only used pg analogues for the entire duration of their treatment. there is limited data on the effects of pg analogues on the corneal microstructure. in a scanning laser confocal microscopic study by bergonzi., pg analogue therapy was found to be associated with increased keratocyte densities in all corneal layers of patients with poag independent of duration of pg analogue treatment. they hypothesized decreased extracellular matrix synthesis secondary to mmp activation with resultant increased keratocyte density within the same region as an explanation for their findings though cct measurements were not made and this hypothesis was not tested in the study. in another study by baratz., pg analogues, -adrenergic blockers, and carbonic anhydrase inhibitors had no discernible effect on either keratocyte or endothelial densities of ocular hypertensive patients treated for a period of 6 years. of note, the study by baratz. did not have a separate study group consisting of glaucoma subjects. in prior reports, pg analogues have been also found to be associated with reduction in tear break - up time and basal tear secretion, increased impression cytology grade of conjunctiva and altered meibomian gland function such as increased meibum score and meiboscore and tear film instability. however, it is yet to be determined whether decreased tear break up time and tear secretion can lead to reduced keratocyte counts in glaucoma patients. the relationship between prolonged treatment with pg analogues and its impact on cct is still unclear. the ocular hypertension treatment study (ohts) group reported that the rate of cct reduction in ocular hypertensive patients treated with topical pg analogues over 3.8 years was more than those who were on beta - blockers. ohts group suggested that the use of topical pg analogues may be associated with a slightly higher rate of thinning in cct. viestenz., performed a nonrandomized controlled cross - sectional study of cct among patients using topical pg analogues and found significantly thinner cct among patients using pg analogue than among controls (539 m vs. 539 m). in a prospective study by sen., a significant reduction in cct at 6, 12, and 24 months was observed compared to baseline levels in both poag and patients with pseudoexfoliation glaucoma treated with pg analogues. in another prospective study by harasymowycz., the effect of topical travoprost on cct was evaluated, and they found a mean decrease in cct of 6.9 m after 6 weeks of treatment. this amount of decline was found to be similar to the 6 month decline with bimatoprost and 12 month decline with latanoprost in the study by sen. reported a significant reduction in cct during a mean treatment interval of 17.9 15.7 months after initial diagnosis of patients with poag and normal tension glaucoma who were treated with topical pg analogues. in an experimental animal model, park,. showed corneal thinning with decreased collagen type i expression in the stroma by immunohistochemical staining in rabbits treated with pg analogues. in the same study, the topical use of pg analogues also resulted in increased mmp to timp ratio, which may be triggered by inflammatory cytokines such as interleukin-1 (il-1) and il-6. in an experimental study by wu., they showed that the latanoprost induced morphological and biomechanical changes in cultured corneal stromal cells and the extracellular matrix, such as modifying type i collagen distribution. however, liu. found that latanoprost had no effect on collagen degradation by corneal fibroblasts. in a prospective study by honda., an increase in mmp-1 and mmp-9 and a decrease in tissue inhibitors of metalloproteinases -1 in tears collected from glaucoma patients who were on pg analogues was detected. it has been shown that pg analogues increase the release of mmps from tenon fibroblasts and ciliary smooth muscle cells. overall, these observations suggest that pg analogues may promote collagen degradation in the stroma. in contrast to the findings of previous studies, a prospective study on medically treated chronic open - angle glaucoma patients reported an increase in cct in patients who received bimatoprost or latanoprost, but no changes in travoprost group during a follow - up period of 2 years. the authors of that study were not able to associate their observations with any causal factor. although our data suggests reduction of cct and keratocyte density with pg analogue therapy, we can not make a distinction whether the observed changes are directly due to pg analogues or the preservative in the medication (benzalkonium chloride [bak ]). although bak exposure has been associated with ocular surface disease, corneal epithelial loss and trabecular meshwork damage, to the best of our knowledge there is no data linking bak to keratocyte loss. however, in a study by martone., higher number of activated stromal keratocytes was observed in the corneas of glaucoma patients who were treated with preservative containing anti - glaucoma drops as compared to those patients on nonpreservative medication, suggesting a possible role of preservatives in stromal changes. one limitation to this study is the lack of baseline cct measurements and keratocyte densities prior to initiation of treatment. cct measurements and ivcm assessments were performed at least 2 years after the initiation of treatment with pg analogues. thus, our data, as well as those obtained in other similar previous studies can not be used to make assumptions on time dependent changes related to pg use in corneas of glaucoma subjects. we also did not have any data on pg drop use in nonglaucomatous (i.e. ocular hypertensive) subjects. ideally, it would have been best to include a nontreated poag group for comparison ; only then we would be able to attribute changes in keratocyte densities to the treatment. however, withholding treatment from glaucoma patients for any period is not possible on ethical grounds. since any class of topical anti - glaucoma medication as well as drop preservatives could adversely affect keratocyte densities, inclusion of a poag group receiving another class of medication would not have made any significant contribution to the interpretation of the keratocyte density reductions. thus, prospective cohort studies with long follow - up intervals are required for to make causal inferences and to obtain longitudinal cct and keratocyte data from a well - studied cohort of individuals with who were on pg analogue treatment. in conclusion, the findings of this study suggest that the long - term treatment with pg analogues may be associated with reduced keratocyte densities and corneal thickness. prospective studies looking into time - dependent alterations of keratocyte densities associated with pg analogue use in both glaucomatous and ocular hypertensive subjects is needed to clarify the exact relationship between topical pg analogue use and its effects on corneal stroma. | purpose : to evaluate whether prostaglandin (pg) analogue use is associated with alterations in keratocyte density and central corneal thickness (cct) in subjects with primary open - angle glaucoma (poag).materials and methods : thirty - five poag patients treated with pg analogues for > 2 years and 35 control subjects without glaucoma were included in this cross - sectional study. all subjects were underwent cct measurements using ultrasound pachymetry. keratocyte densities of each stromal layer were determined by in vivo confocal microscopy. student 's t - test and chi - square test were used for statistical evaluations. correlations between keratocyte densities and cct were analyzed using pearson 's correlation analysis.results:keratocyte densities in each stromal layer were significantly lower in glaucoma patients receiving pg analogues as compared to those of controls (p < 0.001). the mean cct was also lower in glaucoma patients (515.2 18.8) than control subjects (549.6 21.1, p < 0.001). a positive correlation between keratocyte densities in each stromal layer and cct was observed in poag patients.conclusions:long-term administration of topical pg analogues may adversely influence keratocyte densities and cct. further prospective studies are required clarify the relationship between pg analogues and their effects on the cornea. |
whether pelvic and para - aortic lymphadenectomy is of therapeutic benefit in advanced ovarian cancer will remain unclear until the publication of the arbeitsgemeinschaft gynkologische onkologie lymphadenectomy in ovarian neoplasms (ago lion) trial. in early ovarian cancer complete systematic lymphadenectomy is accompanied by morbidity which may be reduced by sentinel node biopsy already established for several solid tumors. in ovarian cancer there are 2 main pathways in lymphatic drainage : along the ovarian vessels to the para - aortic nodes and the uterine vessels to the iliac lymph compartments. following injection of radioactive dye into the ovarian ligaments this could be confirmed suggesting that there is bidirectional flow at this level of the ovarian and uterine lymphatic pathways. indocyanine - green - guided (icg) injection to the uterine corpus seems to be equally effective in labelling the uterine mllerian and the ovarian mesonephric lymphatic drainage of the ovary. this technique was applied and will be outlined in the video showing the procedure with respect to the para - aortic lymphatic drainage. isolated sentinel node biopsy and tumor excision will not resect the organ compartment together with its super - ordinated draining lymphatic system at risk. thus, the authors suggest to remove the malignancy together with its draining lymphatic vessels and at least the first 2 sentinel nodes in each channel en bloc ; we propose to analyze this procedure consistent with the ontogenetic approach with respect to diagnostic accuracy and loco - regional control. | objectivewhether pelvic and para - aortic lymphadenectomy is of therapeutic benefit in advanced ovarian cancer will remain unclear until the publication of the arbeitsgemeinschaft gynkologische onkologie lymphadenectomy in ovarian neoplasms (ago lion) trial. in early ovarian cancer, however, lymphadenectomy seems mandatory for diagnostic and also therapeutic reasons [123].methodscomplete systematic lymphadenectomy is accompanied by morbidity which may be reduced by sentinel node biopsy already established for several solid tumors [456 ]. in ovarian cancer there are 2 main pathways in lymphatic drainage : along the ovarian vessels to the para - aortic nodes and the uterine vessels to the iliac lymph compartments [7 ]. following injection of radioactive dye into the ovarian ligaments this could be confirmed suggesting that there is bidirectional flow at this level of the ovarian and uterine lymphatic pathways [8 ]. indocyanine - green - guided (icg) injection to the uterine corpus seems to be equally effective in labelling the uterine mllerian and the ovarian mesonephric lymphatic drainage of the ovary [910].resultsthis technique [9 ] was applied and will be outlined in the video showing the procedure with respect to the para - aortic lymphatic drainage. isolated sentinel node biopsy and tumor excision will not resect the organ compartment together with its super - ordinated draining lymphatic system at risk.conclusionthus, the authors suggest to remove the malignancy together with its draining lymphatic vessels and at least the first 2 sentinel nodes in each channel en bloc ; we propose to analyze this procedure consistent with the ontogenetic approach [1112 ] with respect to diagnostic accuracy and loco - regional control. this could potentially avoid most of systematic lymphadenectomies in early ovarian cancer. |
with wide application of percutaneous coronary intervention (pci) technology in patients with coronary artery disease (cad), contrast - induced acute kidney injury (ci - aki) has become a serious complication and is the third leading cause of aki in hospitalized patients. in the past several years, the reported incidences of ci - aki ranged from 2% to 30% due to different populations and ci - aki definitions. 's study showed that ci - aki was a significant incremental predictor of cardiovascular events at each stage of chronic kidney disease (ckd) in acute coronary syndrome (acs) patients. emergency procedure was reported to be an independent risk factor of ci - aki, and ci - aki rate in emergency pci patients is significantly higher than in those undergoing selective intervention. however, previous studies of ci - aki were mostly based on data from selective cases. to those undergoing emergency pci, the estimation of the risk of ci - aki is always limited for being pressed for time after admission, and so far, the risk factor profile of ci - aki in emergency pci patients is still unclear and needed to investigate. the aim of this study was to explore the risk factors of ci - aki in a chinese population undergoing emergency pci. from january 1, 2013, to june 30, 2015, patients who underwent emergency pci at a single center (fuwai hospital, beijing, china) were enrolled consecutively. the inclusion criterion was patients undergoing emergency pci ; the exclusion criteria were those (1) who contact with contrast medium 200 ml showed no significant difference between ci - aki and non - ci - aki groups. baseline characteristics and laboratory determinations of ci - aki and non - ci - aki groups data are expressed as n (%) or median (interquartile range). ci - aki : contrast - induced acute kidney injury ; bsa : body surface area ; dm : diabetes mellitus ; mi : myocardial infarction ; tia : transient ischemia attack ; sbp : systolic blood pressure ; dbp : diastolic blood pressure ; lvef : left ventricular ejection fraction ; wbc : white blood cell ; hb : hemoglobin ; hdl - c : high - density lipoprotein cholesterol ; ldl - c : low - density lipoprotein cholesterol ; hs - crp : high - sensitive c - reactive protein ; egfr : estimated glomerular filtration rate. procedural characteristics and medication administration of ci - aki and non - ci - aki groups data are expressed as n (%) or median (interquartile range). ci - aki : contrast - induced acute kidney injury ; iabp : intro - aortic balloon pump ; lad : left anterior descending ; acei : angiotensin - converting enzyme inhibitor ; arb : angiotensin ii receptor blocker ; ccb : calcium channel blocker. univariate analysis for risk factors of ci - aki in emergency pci patients is shown in table 3. a total of 23 variables were analyzed and 15 variables showed significant association with ci - aki. the significant correlates included demographics (female [p 200 ml showed no significant difference between ci - aki and non - ci - aki groups. baseline characteristics and laboratory determinations of ci - aki and non - ci - aki groups data are expressed as n (%) or median (interquartile range). ci - aki : contrast - induced acute kidney injury ; bsa : body surface area ; dm : diabetes mellitus ; mi : myocardial infarction ; tia : transient ischemia attack ; sbp : systolic blood pressure ; dbp : diastolic blood pressure ; lvef : left ventricular ejection fraction ; wbc : white blood cell ; hb : hemoglobin ; hdl - c : high - density lipoprotein cholesterol ; ldl - c : low - density lipoprotein cholesterol ; hs - crp : high - sensitive c - reactive protein ; egfr : estimated glomerular filtration rate. procedural characteristics and medication administration of ci - aki and non - ci - aki groups data are expressed as n (%) or median (interquartile range). ci - aki : contrast - induced acute kidney injury ; iabp : intro - aortic balloon pump ; lad : left anterior descending ; acei : angiotensin - converting enzyme inhibitor ; arb : angiotensin ii receptor blocker ; ccb : calcium channel blocker. univariate analysis for risk factors of ci - aki in emergency pci patients is shown in table 3. a total of 23 variables were analyzed and 15 variables showed significant association with ci - aki. the significant correlates included demographics (female [p 200 ml in the body of the literature which was due to the popularization of nonionic, iso - osmolar contrast medium and the lowest volume used during intervention procedure in recent years, bsa, representing the area where drug distributes, showed the toxicity intensity of contrast medium. in other words, bsa was an alternate of cv in the risk factor profile of ci - aki in emergency procedure population in the study. as for the periprocedural medication, the administration of aspirin could reduce the risk of ci - aki from the study though only three and one patient did not contact aspirin in ci - aki and non - ci - aki group, respectively, supporting the hypothesis that contrast medium could induce thrombosis in the development of ci - aki. 's study also clarified that administration of heparin before procedure could significantly decline the rate of ci - aki, indicating that additional antithrombotic treatment may matter a lot in ci - aki prevention. however, the use of diuretics could increase the risk of ci - aki, similar to solomon. the occurrence of ci - aki is related to toxic effect of contrast medium on the tubular epithelial cells and results directly from hemodynamic disturbances of the renal blood flow. the pre- or post - procedural use of diuretics can directly lead to ci - aki through reducing renal blood flow and enhancing the toxicity of contrast medium due to blood concentration. by contrast, periprocedural hydration, as the cornerstone of ci - aki prevention, can help reverse the negative hemodynamic conditions in clinical practice. in the body of the literature, a higher egfr served as a promoting factor in the study showing contrary performance to prior reports, which might be attributed to high sensitivity of ci - aki definition and special nature of the study population. a higher egfr usually came from a relatively lower scr correspondingly and was apt to be diagnosed as ci - aki for a slight fluctuation but scr increases 25% above baseline. consequently, egfr showed the illusion of positive correlation with ci - aki through ckd - epi formula conversion. besides, preprocedural statin contact showed no statistical significance in the present study, while several previous studies concluded that statin therapy was effective in reducing the risk of ci - aki in acs patients owing to pleiotropic effects (the anti - inflammatory, anti - apoptotic, and anti - thrombotic properties) with few side effects. the probable reasons of contrary results might be attributed to the paucity of high - dose statin cases, small duration of statin contact before procedure, and diversification of statin administration in the real word. in future, large and well - designed studies are needed to confirm the preventive effect of preoperative statin therapy and determine the rational dose and timing in emergency patients. above all, comparing to prior studies, low bsa and diuretics use are two new risk factors of ci - aki in our study. moreover, some identified risk factors in previous reports are also present in our research, for example, low lvef, hb level, and lad stented. whereas, some other variables, such as age, female, hypertension, diabetes mellitus, peripheral vascular disease, iabp application, and cv, were reported to be independent risk factors of ci - aki in selective pci cases, showing no significance in our study. hence, different kinds of patients should deserve different risk factor profile to achieve the most accurate estimation of the risk of ci - aki clinically. first, the present study was based on patients enrolled from a single center and the data were collected retrospectively. therefore, our results are subjected to limitations inherent to the observational nature of a retrospectively collected database. second, due to the regular application of periprocedural hydration, we could not confirm the concrete and specific influence exerted on the true baseline scr before intervention. third, though the definition of ci - aki in the present study is used universally in previous reports, it might not be suitable for emergency population according to egfr 's abnormal performance. a more scientific and appropriate critical value of scr increase in the definition of ci - aki in an emergency pci population should be explored in future. this study demonstrated that the incidence of ci - aki in emergency pci patients was high. history of mi, low bsa, lvef and hb level, lad stented, and diuretics use are associated with increased risk of ci - aki in patients undergoing emergency pci. | background : previous studies of contrast - induced acute kidney injury (ci - aki) were mostly based on selective percutaneous coronary intervention (pci) cases, and risk factors of ci - aki after emergency pci are unclear. the aim of this study was to explore the risk factors of ci - aki in a chinese population undergoing emergency pci.methods:a total of 1061 consecutive patients undergoing emergency pci during january 2013 and june 2015 were enrolled and divided into ci - aki and non - ci - aki group. univariable and multivariable analyses were used to identify the risk factors of ci - aki in emergency pci patients. ci - aki was defined as an increase in serum creatinine 25% or 0.5 mg / dl (44.2 mol / l) above baseline within 3 days after exposure to contrast medium.results:the incidence of ci - aki in patients undergoing emergency pci was 22.7% (241/1061). logistic multivariable analysis showed that body surface area (bsa) (odds ratio [or ] 0.213, 95% confidence interval [ci ] : 0.0750.607, p = 0.004), history of myocardial infarction (mi) (or 1.642, 95% ci : 1.0792.499, p = 0.021), left ventricular ejection fraction (lvef) (or 0.969, 95% ci : 0.9440.994, p = 0.015), hemoglobin (hb) (or 0.988, 95% ci : 0.9761.000, p = 0.045), estimated glomerular filtration rate (or 1.027, 95% ci : 1.0181.037, p < 0.001), left anterior descending (lad) stented (or 1.464, 95% ci : 1.0002.145, p = 0.050), aspirin (or 0.097, 95%ci : 0.0090.987, p = 0.049), and diuretics use (or 1.850, 95% ci : 1.2332.777, p = 0.003) were independent predictors of ci - aki in patients undergoing emergency pci.conclusion:history of mi, low bsa, lvef and hb level, lad stented, and diuretics use are associated with increased risk of ci - aki in patients undergoing emergency pci. |
insulin resistance and systemic inflammation frequently occur in infants undergoing cardiac surgery with cardiopulmonary bypass (cpb). insulin resistance presenting with increased blood glucose level (hyperglycemia) and decreased sensitivity to insulin increases morbidity and mortality in critically ill patients [1, 2 ]. intensive insulin therapy aiming at euglycemia improves their clinical outcome [35 ]. in a recently published study involving patients undergoing cardiac surgery, intraoperative insulin resistance was associated with an increased risk of short - term adverse outcomes. the inflammatory reaction and injury may contribute to the development of postoperative complications [7, 8 ]. the magnitude and duration of the systemic inflammatory response determine the development of tissue damage, multiorgan failure, or even death [9, 10 ]. our previous studies have demonstrated that ameliorating insulin resistance attenuates the systemic inflammatory response in infants undergoing cpb. adiponectin, a hormone derived from the adipose tissue, has been demonstrated to have insulin - sensitizing and anti - inflammatory properties in obesity and type 2 diabetes mellitus. recently adiponectin has also been shown to have a reverse correlation with insulin resistance and inflammatory mediators. studies on the relationship of adiponectin with insulin resistance and inflammatory mediators in infants undergoing cardiac surgery with cardiopulmonary bypass are scarce. the present study was undertaken to investigate the association of adiponectin with the development of insulin resistance and kinetic changes of inflammatory mediators in infants undergoing cpb. the present study has been approved by the ethics committee of xijing hospital, the fourth military medical university, and performed according to the world medical association declaration of helsinki. patient population : infants aged between 6 months and 3 years undergoing open cardiac surgery with cpb for congenital heart disease were enrolled for the study at our hospital from june 2010 to august 2011. exclusive criteria included preoperative liver and kidney disease or dysfunction, preoperative coagulation disorder, palliative or second operation, and impaired blood glucose levels. blood glucose was monitored on an hourly basis and insulin infusion rate was adjusted to maintain glucose levels between 4.4 and 8.3 mmol / l. the infusion of insulin is a standard of care and started when the glucose concentration became higher than 8.3 mmol / l. an insulin glycaemic index (insulin glucose/22.5) was calculated at each time point. blood samples were taken at 7 time points for each patient as follows : before anesthesia (t1), at the initiation of cpb (t2), at the termination of cpb (t3), 6 h after cpb (t4), 12 h after cpb (t5), 24 h after cpb (t6), and 48 h after cpb (t7). serum level of adiponectin was determined with a commercial enzyme - linked immunosorbent assay (r&d, wiesbaden, germany). serum insulin levels were measured with an insulin kit (r&d systems, abingdon, uk). plasma il-6 and tnf- levels were determined using commercially available elisa kits (r&d systems, abingdon, uk). all enzyme - linked immunosorbent assay (elisa) protocols were carried out according to kit guidelines. pearson 's correlation coefficient was estimated for associations between adiponectin and metabolic variables at different time points. repeated measures analysis of variance (anova) models (figures 1, 2, and 3) were analysed using spss version 13.0 (spss, inc., baseline characteristics of the study participants are shown in table 1. the cardiac surgery included repair of ventricular septal defects in 35 patients, atrial septal defects in 18 patients, and correction of tetralogy of fallot in 7 patients. serum insulin concentrations increased at the termination of cpb, following the course of exogenously applied insulin, and remained stable thereafter (figure 1(b)). to create a more specific parameter of insulin resistance that combines serum glucose with serum insulin levels, we calculated an insulin glycaemic index (insulin glucose/22.5) at each time point (figure 1(c)). the insulin glycaemic index increased during the first 22 hours of the observation period and remained stable thereafter reflecting the kinetics of exogenously applied insulin. during the observation period inflammatory cytokines rapidly increased with peak concentrations of tnf- and il-6 at the 6 h time point (figures 2(a) and 2(b)). adiponectin serum levels were repressed throughout the observation period reaching a minimum at the 6 h time point (figure 3). there was no association between the adiponectin at t3, t5, t6, and t7 time points and glycemic index, tnf - alpha and il-6 (table 2). at t4 (6 h after cpb) we found significant inverse correlations of adiponectin with insulin glycaemic index, il-6, and tnf- (figure 4). correlation of adiponectin with the insulin glycaemic index was r = 0.465 (p < 0.001) was adiponectin with il-6, r = 0.427 (p < 0.001), and adiponectin with tnf- was r = 0.447 (p < 0.001). several studies have reported that adiponectin has a negative correlation with insulin resistance in chronic diseases such as metabolic syndrome and type 2 diabetes [15, 16 ]. however, the relationship of adiponectin with insulin resistance and inflammatory mediators in infants undergoing cardiac surgery with cardiopulmonary bypass has not been identified so far. the present study demonstrated the correlation of adiponectin with insulin resistance and the kinetic changes of inflammatory cytokines in infants undergoing cpb. the marked increases in the amount of exogenous insulin requirement to maintain euglycemia as well as circulating insulin levels during cpb surgery suggest the development of insulin resistance. our study showed significant increase in tnf- and il-6 levels after the initiation of cpb and their kinetics at various time points. at the same time, the need of an increased rate of insulin infusion to maintain euglycemia following the operation suggested the development of insulin resistance. insulin resistance is associated with the inflammatory response, but its molecular basis and physiological significance are not fully understood. inflammatory mediators such as tnf- and il-6 either alone or through synergistic effect could lead to the development of insulin resistance by blocking the signal transduction of insulin, impairing insulin sensitivity, and increasing free fatty acids [17, 18 ]. adiponectin has been shown to directly or indirectly affect insulin sensitivity through modulation of insulin signaling and the molecules involved in glucose and lipid metabolism. adiponectin - deficient mice were shown to be prone to diet - induced obesity and insulin resistance and its reversal by adiponectin treatment. in humans, low adiponectin was more closely associated with insulin resistance than adiposity. in infants undergoing cardiac surgery, il-6 and tnf- levels were markedly increased while serum adiponectin levels were moderately decreased. this suggests the inverse relationship of circulating adiponectin levels to il-6 and tnf- and insulin resistance in critically ill patients. the repression of adiponectin serum levels in our model and its association with insulin resistance are in agreement with previous reports [13, 21 ]. this indicates the role of adiponectin in regulation of glucose metabolism (insulin resistance) and inflammatory mediators. in summary, we have demonstrated the significant inverse association of adiponectin with markers of systemic inflammation and insulin resistance in infants undergoing open cardiac surgery. the better understanding of the association of adiponectin with insulin resistance and systemic inflammation will be of high clinical value as it may have therapeutic implications. | background. insulin resistance and systemic inflammation frequently occur in infants undergoing cardiac surgery with cardiopulmonary bypass, while adiponectin has been demonstrated to have insulin - sensitizing and anti - inflammatory properties in obesity and type 2 diabetes mellitus. in this prospective study, we aimed to investigate the association of adiponectin with insulin resistance and inflammatory mediators in infants undergoing cardiac surgery with cardiopulmonary bypass. methods and results. from sixty infants undergoing open cardiac surgery, blood samples were taken before anesthesia, at the initiation of cardiopulmonary bypass and at 0, 6, 12, 24, and 48 hours after the termination of cardiopulmonary bypass. plasma interleukin-6 (il-6), tumor necrosis factor - alpha (tnf-), and adiponectin levels were assessed in blood samples. insulin resistance was measured by assessment of the insulin requirement to maintain euglycaemia and repeated measurements of an insulin glycaemic index. insulin glycaemic index, il-6, and tnf- increased up to 38-fold 6 h after the operation. adiponectin is negatively correlated with markers of systemic inflammation 6 h after cpb. conclusions. although the level of serum adiponectin decreased significantly, there was a significant inverse association of adiponectin with markers of systemic inflammation and insulin resistance in infants undergoing open cardiac surgery. |
patient - doctor relationship has been defined as a meaningful therapeutic interaction between the patients and their doctors and is considered as the seventh element of healthcare quality. meaningful therapeutic interaction between patients and doctors includes both patient - doctor communication and trust. an effective patient - doctor communication consists of explaining patient 's illnesses and treatments, spending adequate time, managing uncertainty, and developing rapport [35 ]. patient - doctor trust is high when the doctor is technically competent, is thorough and careful in examining the patient, has good listening capabilities, and has complete understanding of patient 's illnesses and treatments [610 ]. effective communication has been found to enhance patient 's engagement in care, satisfaction with care, and treatment adherence. effective communication has also been found to improve health outcomes [14, 15 ]. among older patients with diabetes, patient - doctor communication has been found to be associated with lower blood pressure, lower anxiety, and better quality of life. similarly, high trust in the doctor has been found to be associated with lower hassles in self - care, better ability to perform diabetes care activities, and satisfaction with the doctor. effective patient - doctor communication and trust become especially important in managing patients with two or more chronic conditions (i.e., multimorbidity). multimorbidity affects elderly population disproportionately and is projected to increase from 34,835 million in 2000 to 81,999 million by 2050. elderly individuals with multimorbidity account for 98% of hospital readmissions, incur 93% of total medicare spending, and experience higher rates of preventable hospitalizations as compared to those without multimorbidity. multimorbidity also affects care coordination and leads to poor management of cooccurring conditions due to the focus on one dominant chronic condition. therefore, policymakers, providers, and researchers have emphasized the need for meaningful interactions between patients and doctors in managing the burden of multimorbidity [2326 ]. an effective communication and high trust with the doctor can improve the care of the elderly with multimorbidity. a special case of multimorbidity presence of mental health condition in addition to chronic physical conditions can be very challenging for disease management [2730 ]. effective communication and trust have been shown to improve outcomes for not only individuals with chronic physical conditions but also those with a mental health condition. for example, effective communication decreased the patient health questionnaire (phq-9) scores among adults with depression from baseline to one - month follow - up, indicating an improvement in the severity of depression. however, a survey sponsored by the national depressive and manic - depressive association (dmda) found that ineffective communication exists between primary care physicians and patients with depression. for example, the survey found that even though doctors thought that they explained the potential adverse effects of antidepressant medications, the patients felt a lack of information on certain adverse effects such as sexual dysfunction, weight gain, and insomnia. also, trust in the doctor is important for patients with mental illness to maintain the confidentiality of their mental illness due to its associated social and personal stigma. there is a lack of research on the impact of having a mental health condition in addition to multiple chronic physical illnesses on communication and trust with the doctor among elderly individuals. we could only find two studies (one in the us and one in germany), which evaluated the relationship between number of chronic conditions and patient - doctor relationship [35, 36 ]. in the us, a study among noninstitutionalized adults aged 18 years or older from 12 metropolitan communities analyzed the relationship between number of chronic conditions and scores on patient - doctor communication scale. the study found a small but significant relationship between higher number of chronic conditions and ineffective patient - doctor communication. another study among the german adults examined the factors associated with patient - doctor relationship which was measured using the german version of the patient - doctor relationship questionnaire (pdrq-9). the study found that individuals with lower number of chronic physical and mental comorbidities reported higher scores on the pdrq-9, indicating the higher quality of patient - doctor relationship. therefore, the primary objective of this study is to examine patient - doctor communication and trust among the elderly who had both chronic mental and physical conditions as compared to those having multiple physical conditions without any mental condition. we hypothesized that elderly patients having both mental and physical conditions will be more likely to have ineffective communication and have low trust with the doctor as compared to those having multiple physical conditions without any mental health condition. according to the previous studies, factors other than multimorbidity can affect meaningful therapeutic interactions between the patients and their doctors. these may include age, gender, race / ethnicity [37, 38 ], socioeconomic status, health status, and length of the visit. therefore, we utilized the theoretical model of patient - doctor relationship by gabay (2015) to guide the selection of independent variables used in our study. according to this model, patient - doctor relationship is influenced by (a) sociodemographic factors such as patient 's age, race / ethnicity, gender, education, insurance, and metropolitan area ; (b) health condition factors including multimorbidity and body mass index ; (c) health status such as perceived general health status and functional health status ; and (d) patient 's satisfaction with care. we adopted a cross - sectional study design using data from a nationally representative survey of medicare beneficiaries. we utilized data from the access to care module of the medicare current beneficiary survey (mcbs) 2012. the mcbs is a continuous multipurpose survey of a nationally representative sample of medicare beneficiaries conducted by the centers for medicare & medicaid services [42, 43 ]. the mcbs is conducted among all medicare beneficiaries who are above 65 years of age or who are disabled. the survey is conducted through computer - assisted personal interviews (capi) with the beneficiaries. the person level survey data is released in two modules : access to care (ac) and cost and use (cu). we utilized the ac module of the mcbs that uses valid and reliable survey questions to assess patients ' experiences with care received from their provider. the ac module represents the population of individuals who were always enrolled in the medicare during the calendar year. it contains deidentified information on each participant 's access to health care, usual source of care, and satisfaction with care. the ac module also includes demographic, health insurance, health status, and functioning data. the mcbs is a longitudinal panel survey, where individuals are interviewed three times a year for a maximum of four years. the participants are selected from the medicare enrollment file using a multistage and stratified sampling design to be representative of the medicare population. the multistage and stratified sampling design is commonly used in large national surveys to feasibly conduct survey, reduce bias, and produce nationally representative survey results. in the multistage design it avoids the sampling of all the units which can be expensive and time consuming. in stratified sampling design, the beneficiaries are divided into seven strata based on age : 0 to 44, 45 to 64, 65 to 69, 70 to 74, 75 to 79, 80 to 84, and 85 or older. the sampling units are then selected from each stratum so that the results can be representative of the total population. we included community - dwelling elderly medicare beneficiaries who were aged 65 years or older, had at least one chronic physical condition, and had a usual source of care in our study. yes to the question is there a particular medical person or a clinic you usually go to when you are sick or for advice about your health ? further, we excluded the individuals with missing data for chronic health conditions, communication, or trust variables. we utilized the participants ' responses to the survey questions from 2012 ac module of the mcbs in this study. a survey item in the mcbs asked about the specialty of participants ' doctor. about 90% of beneficiaries answered family practice, general practice, or internal medicine. thus, most beneficiaries referred to their primary care physician while responding to the survey items about communication and trust with their doctor. an effective patient - doctor communication consists of discussing patient 's illness and treatment, spending adequate time, building rapport, and managing patient 's uncertainty [3, 5 ]. we used the following six survey items from the mcbs which assessed these components of communication with the doctor to construct a patient - doctor communication scale (table 1) : (a) your doctor tells you all you want to know about your condition and treatment, (b) your doctor often does not explain your medical problems to you, (c) you often have health problems that should be discussed but are not, (d) your doctor often seems to be in a hurry, (e) your doctor often acts as though he was doing you a favor by talking to you, and (f) your doctor answers all questions. the response scale ranged from one to four (1 : strongly agree, 2 : agree, 3 : disagree, and 4 : strongly disagree). we reverse coded the survey items (a) and (f) to make their scores consistent with other items. the sum of scores of these six survey items ranged from 6 to 24 and was highly skewed. thus, we dichotomized the total score on communication scale by using median split method to create a binary response variable. the elderly with total communication score equal to or above the median value (18) were categorized as having effective communication with the doctor while those with scores below median value were considered to experience ineffective communication. patient 's trust in the doctor is influenced by technical skills of the doctor, thorough examination of the patient, and complete understanding of patient 's medical conditions [610 ]. we utilized the following five survey items from the mcbs which measured these components of trust to construct patient - doctor trust scale : (a) your doctor is very careful to check everything when examining you, (b) your doctor is competent and well - trained, (c) your doctor has a good understanding of your medical history, (d) your doctor has a complete understanding of the things that are wrong with you, and (e) you have great confidence in your doctor. the response scale ranged from one to four (1 : strongly agree, 2 : agree, 3 : disagree, and 4 : strongly disagree). the sum of scores of these five survey items ranged from 5 to 20 and was highly skewed. hence, we dichotomized the total score on trust scale by using median split method to create a binary response variable. the elderly with a total score equal to or above median value (17) were categorized as having high trust in the doctor while those with scores below median value were considered to have low trust in the doctor. we utilized participants ' having been told by a doctor that they have a chronic condition to assess the presence of multimorbidity. we selected chronic conditions based on the conceptual framework by goodman and colleagues for defining and measuring chronic conditions for research, policy, program, and practice. the chronic physical conditions included arthritis (osteoarthritis or rheumatoid arthritis), cancer (skin or any other cancer), diabetes (type 1 or type 2), heart disease (angina pectoris, myocardial infarction, or any other heart condition), hypertension, osteoporosis, and respiratory diseases (asthma, chronic obstructive pulmonary disease, or emphysema). the mental health conditions consisted of depression or any other mental or psychiatric disorder. based on the presence of these chronic physical and mental health conditions, we defined three multimorbidity categories : (a) no mm (no multimorbidity, that is, only one physical condition), (b) mm - pi (two or more physical conditions but no mental illness), and (c) mm - pi&mi (both physical and mental health conditions). we also included patient 's body mass index (underweight / normal, overweight, and obese / morbid obese) as a health condition factor. the sociodemographic factors included beneficiaries ' age, sex, race / ethnicity, marital status, education level, annual personal income, supplemental insurance (medicaid) and type of insurance (hmo / ffs), and external environmental characteristics (metro status and region of residence). we measured patient 's self - perceived general health status as compared to others of the same age (excellent / very good, good, or fair / poor). we also measured functional health status by examining participants ' self - reported difficulty with activities of daily living (adl) including difficulty in walking, eating, bathing, dressing, getting in or out of bed or chair, or using the toilet [4750 ]. we classified participants ' difficulty with adl in three categories : (a) no adl, (b) 1 - 2 adl, and (c) 36 adl. patient 's satisfaction with care constitutes an important factor which can affect patient - doctor communication and trust. we used participants ' responses to the survey item please tell me how satisfied you have been with the overall quality of the health care you have received over the past year to assess patient 's satisfaction with care [5154 ]. dissatisfied or very dissatisfied were considered dissatisfied with the quality of care. we analyzed the validity of patient - doctor communication and trust scales using factor structure by the principal component analysis with promax rotation and internal consistency (a measure of reliability) by cronbach 's alpha coefficient. the principal component analysis yielded single factor for both patient - doctor communication and trust scales. the single factor accounted for 98% of variance for the communication scale and 100% of variance for the trust scale. the alpha coefficient value for the communication scale was 0.87 and for the trust scale was 0.92, which indicated high reliability for both the scales. we used chi - square tests to examine the unadjusted associations of patient - doctor communication and trust with theoretically derived predictor variables (e.g., patient 's sociodemographic characteristics, health status, health condition, and patient 's satisfaction factors). then, correlations were used to assess multicollinearity, resulting in the removal of patient 's health status (self - perceived general health and functional status) from further analysis. multivariable logistic regressions were conducted to examine the relationship between multimorbidity and patient - doctor relationships, after adjusting for patient 's sociodemographic factors, health condition, and satisfaction with care. we used survey procedures with statistical analysis system (sas) software to account for the complex survey design. among 9,867 medicare beneficiaries included in this study (table 1), the majority were white (78.5%), lived in a metro region (77.4%), did not have limitations in activities of daily living (66.2%), were either overweight or obese (64.6%), and were satisfied with quality of care (97.4%). also, 56.6% elderly beneficiaries were women, 57.2% were married, 45.4% had more than high school education, 35.1% were enrolled in hmo, 12.3% had medicaid insurance, 19.6% had fair or poor general health status, and 58.6% were past or current smokers (table 1). more than two - thirds of beneficiaries had mm - pi (72.0%) and 12.1% had mm - pi&mi, constituting 84.1% of beneficiaries with either type of multimorbidity. from the unadjusted analysis, mm - pi&mi was present among a higher percentage of women than men (14.5% versus 9.0%), current smokers than nonsmokers (20.2% versus 11.3%), those aged 6569 years than 7579 years (14.3% versus 10.6%), those having less than high school education than more than high school education (15.2% versus 10.9%), having $ 50,000 income (16.5% versus 8.5%), and having medicaid insurance (20.6% versus 11.0%) (table 1). an overwhelming majority of the elderly strongly agreed or agreed that their doctor tells them all that they want to know about their condition and treatment (95.7%) and (97.5%). also, a majority of beneficiaries strongly disagreed or disagreed that they often have health problems that should be discussed but are not (92.2%), and often acts as though he was doing them a favor by talking to them a lower percentage of the elderly with mm - pi&mi (80.1%) had effective communication with the doctor as compared to those with mm - pi (84.2%) and no mm (89.7%) (table 3). a majority of beneficiaries agreed or strongly agreed that they have great confidence in their doctor (99.1%), is very careful to check everything when examining them (95.4%), has a good understanding of their medical history (97.7%), and has a complete understanding of the things that are wrong with them (96.6%) (table 2). from the unadjusted analysis, there was no significant difference in trust in the doctor among the elderly with different multimorbidity categories (table 3). from the adjusted multivariate analysis, type of multimorbidity had a significant association with patient - doctor communication. after adjusting for patient 's sociodemographic characteristics, patient 's health condition, and patient 's satisfaction with care, medicare beneficiaries with mm - pi&mi were significantly less likely to have effective communication with the doctor (adjusted odds ratio [95% confidence interval ] = 0.80 [0.68, 0.96 ]), as compared to those with mm - pi (table 4). further, the elderly with no mm were significantly more likely to have effective communication with the doctor (1.48 [1.21, 1.82 ]), as compared to those with mm - pi. other factors significantly associated with effective communication with the doctor were higher education and higher satisfaction with the quality of care. further, those living in the west were less likely to have efficient communication with the doctor as compared to those living in the northeast (table 4). the multimorbidity did not have a significant association with elderly patient 's trust in the doctor (mm - pi&mi versus mm - pi : 0.98 [0.84, 1.15 ]). other variables significantly associated with high trust in the doctor included female sex, higher education, higher personal income, and higher satisfaction with the quality of care (table 4). the elderly above 80 years of age compared to 6569 years, those who are african american or other race compared to white race, and those living in a metropolitan area compared to nonmetropolitan area were less likely to have high trust in the doctor (table 4). with multimorbidity being a norm in the us elderly population, policy focus has recently shifted towards the better illness management of these individuals [58, 59 ]. elderly patients with multimorbidity need care that is responsive to the interactions between multiple conditions and treatments and is tailored to their individual needs. an effective communication and high trust between the patient and doctor are necessary for improving the care of these patients. to our knowledge, this is the first study to examine the association between multimorbidity with or without a mental condition and patient - doctor communication and trust among a nationally representative population of the elderly in the us. our results suggest that a majority of elderly medicare beneficiaries experience effective communication and have high trust in their doctor. however, the elderly who had a mental health condition in addition to chronic physical conditions were less likely to have effective communication as compared to the elderly who had multiple physical conditions without any mental condition. these results are consistent with a previous study that found an ineffective communication between doctor and patient with a single chronic mental condition. the elderly with a mental condition have been found to have different communication needs from individuals without any mental condition. for example, nonverbal communication activities such as gesture and facial activity are impaired among patients with a mental condition. the elderly with both mental and physical chronic conditions may also have social and internal stigma due to mental condition which might affect their communication with the doctor as compared to those with multiple physical conditions without mental condition. our results suggest that clinicians need to pay additional attention for an effective communication and higher trust when treating patients with concurrent physical and mental conditions. nearly fifteen percent of elderly beneficiaries with both chronic physical and mental conditions agreed or strongly agreed that their doctor is often in a hurry. a study by quirk. found that doctor 's appearing to be in a hurry during the visit is perceived as an uncaring attitude by the patients. patients place importance on having a doctor who talks at a slow pace and allows the patient to take his / her time. it is especially critical for an elderly patient with multiple chronic conditions because elderly patients and their families have to take care of all the cooccurring physical and mental health conditions at the same time and may want to talk about all of their diagnoses and comprehend numerous prescribed drugs and treatments. it is important for the clinicians not to hurry during consultation with an elderly patient having multimorbidity and allow the patient to take his / her own time to understand their multiple diagnoses and treatments. multimorbidity presents a challenge to primary care providers for addressing the competing demands of multiple physical and mental conditions in busy outpatient settings. discussion of multiple illnesses and treatments within a short visit may leave some patients feeling that the doctor did not discuss or provide all the information regarding their conditions and treatments, which we observed in our study. physicians who are treating patients with chronic physical and mental conditions may want to explore some alternative solutions to improve the discussion of illnesses and treatments such as the use of printed materials or internet, patient self - care, and use of physician assistants or nurse practitioners in primary care which can improve communication and trust. while we recognize the limitations of self - reported measures of mental illness, it should be noted that survey data has been routinely used for surveillance of mental illness and its associated health and cost burden. also, the mcbs utilizes computer - assisted interview procedures to improve the quality of the survey data. the participants are surveyed at relatively short intervals and the collected information is verified from medical claims to minimize errors. it is possible that patients who reported multiple chronic physical and mental conditions had more critical assessments of their interaction with their provider. also, communication and trust are container concepts and comprise several different domains [33, 66 ]. patients ' perceived communication and trust with the doctor might be influenced by several other factors such as the severity of patient 's chronic condition, differences in patient and providers ' culture and communication styles, and organizational factors. multimorbidity is, therefore, one of the many factors that affect patient - doctor communication and trust. the study sample included elderly individuals who had at least one chronic physical condition. by excluding patients who have no comorbidities, any differences in communication and trust observed among the three groups were likely due to the presence of multimorbidity only. we did not compare patients with and without chronic conditions as this has been covered in the literature. further, we did not use a continuous variable for multimorbidity such as number of chronic conditions because we wanted to examine the association of type of multimorbidity with communication and trust. therefore, we categorized the patients into three groups according to the presence of multiple chronic physical and mental conditions. the strengths of our study include the use of a comprehensive list of survey items from mcbs ac module to measure specific components of patient - doctor communication and trust, which has been lacking in existing studies. we used a nationally representative dataset with a large sample size, and our study results can be generalized to elderly noninstitutionalized medicare beneficiaries throughout the us. also, our study used mcbs, which is a valid multipurpose survey and one of the first to use capi for data quality. the majority of elderly medicare beneficiaries experienced effective communication and high trust with their primary care doctors. the elderly with both chronic physical and mental health conditions had ineffective communication with their doctor as compared to those with multiple physical illnesses only. doctor being often in a hurry and doctor often not explaining illnesses were the two most significant communication issues reported by those with multimorbidity. in the absence of evidence - based clinical guidelines, better patient - doctor communication is required to overcome the challenges of managing multiple chronic conditions. it is important to examine and explain the multiple medical conditions and treatments among patients with multimorbidity. | background. effective communication and high trust with doctor are important to reduce the burden of multimorbidity in the rapidly aging population of the us. however, the association of multimorbidity with patient - doctor communication and trust is unknown. objective. we examined the relationship between multimorbidity and patient - doctor communication and trust among the elderly. method. we used the medicare current beneficiary survey (2012) to analyze the association between multimorbidity and patient - doctor communication and trust with multivariable logistic regressions that controlled for patient 's sociodemographic characteristics, health status, and satisfaction with care. results. most elderly beneficiaries reported effective communication (87.597.5%) and high trust (95.499.1%) with their doctors. the elderly with chronic physical and mental conditions were less likely than those with only physical conditions to report effective communication with their doctor (adjusted odds ratio [95% confidence interval ] = 0.80 [0.68, 0.96 ]). multimorbidity did not have a significant association with patient - doctor trust. conclusions. elderly beneficiaries had high trust in their doctors, which was not affected by the presence of multimorbidity. elderly individuals who had a mental condition in addition to physical conditions were more likely to report ineffective communication. programs to improve patient - doctor communication with patients having cooccurring chronic physical and mental health conditions may be needed. |
buffer solutions are used in biomolecular research to electrostatically stabilize titratable molecular groups such as polar amino acid side chains in proteins and charged lipid head groups. effects of ph buffers on membrane physical properties are generally neglected except in a few recent reports. zwitterionic buffers belonging to good s series were shown to affect the interactions between neighboring membranes and possibly alter membrane bending rigidity. membranes made of phosphatidylcholine (pc) lipids tend to form multilamellar lipid vesicles (mlvs) where the equilibrium repeat spacing (d - spacing) is set by a balance of attractive and repulsive forces. these forces include van der waals (vdw) attraction, hydration repulsion, fluctuation repulsion, and electrostatics. zwitterionic buffers such as 2-(n - morpholino)ethanesulfonic acid (mes), 3-morpholinopropane-1-sulfonic acid (mops), and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (hepes) have been shown to swell mlvs by modifying membrane interactions in at least three ways similar to swelling in salt : (1) reduction of vdw attraction due to dielectric properties, (2) addition of electrostatic repulsions due to binding the lipid water interface, and (3) alteration of membrane bending rigidity possibly by inserting into the lipid bilayer. work presented in this letter shows that this combination of effects can lead to phase coexistence in the case of 1-palmitoyl-2-oleoyl - sn - glycero-3-phosphocholine (popc) bilayers. similar phase coexistence was reported by rappolt. in the presence of alkali chloride salts, notably in the presence of licl. no such phase coexistence was detected for the symmetric shorter - chain dlpc in either monovalent salt or buffer solutions. data presented in this work show that hepes, tris(hydroxymethyl)aminomethane (tris), and ethylenediaminetetraacetic acid (edta), which are standard buffering agents used to stabilize proteins induce phase separation in popc in which the difference in d - spacing between the two phases grows with increasing buffer concentration. specifically, hepes was studied systematically incorporating a range of concentrations that includes those seen in studies, which stabilize membrane proteins in assays in the presence of synthetic lipid systems. in addition, the relative fraction of the two phases correlates with the ph of the buffered solution. hepes has two titratable groups and therefore two pka values : a tertiary amine group (pka1 = 7.5) and a sulfonic acid groups (pka2 = 3.0), which confers a range of electrostatic properties depending on ph. in addition to hepes, typical buffer solutions can also contain other components such as tris, which has a primary amine (pka 7.5) that stabilizes proteins and dna, and chelating agents such as edta, whose two amines (pka 6.1, 10.34) and four carboxylates (pka 0.0, 1.5, 2.0, and 2.7) seek to bind and sequester metal ions such as ca and fe. hence, small - angle x - ray scattering data of popc in the presence of hepes solutions, with and without these other buffering agents, are presented to document their effects on intermembrane interactions. 1-palmitoyl-2-oleoyl - sn - glycero-3-phosphocholine (popc) and 1,2-dilauroyl - sn - glycero-3-phosphocholine (dlpc) were purchased from avanti polar lipids (alabaster, al). all buffer components were purchased from fisher scientific (pittsburgh, pa). conductivity measurements of buffer solutions were conducted using a ge conductivity cell (cat. following previously described methods, mlv liposomes were prepared by hydrating between 10 and 60 mg of lyophilized lipid powder in 1 to 6 ml buffer solution to a final lipid concentration of 10 mm. solutions were put through 3-freeze / thaw cycles and were then allowed to equilibrate at room temperature for more than 3 - 5 days. lipids were tested for sample deterioration by saxs on two different occasions within a 4-month time span and had negligible changes in their scattering profiles. small angle x - ray scattering (saxs) measurements were performed at the advanced photon source beamline 12-id - b and 12-id - c. the energy at beamline 12-id - b was fixed at 14 kev, with data collected using a 2 m pilatus detector set for a sample - detector distance of 1900 mm. the pinhole setup at 12-id - c used a photon energy of 12 kev, using a 4-quadrant mosaic x - ray ccd camera platinum detector built in house (1024 1024 pixel). the sample - detector distance was 2200 mm and had a flux of approximately 5 10 photons / second. lipid samples were x - rayed either in glass capillaries or suspended in droplet form in the x - ray beam path at 23 c for 0.1 s. two - dimensional (2d) scattering data for five shots were averaged and integrated over the angle to obtain intensity versus q (). a custom matlab script was used in fitting lorentzian curves to the center portion of the scattering peaks and to calculate the d - space values. figure 1 shows saxs measurements conducted at ambient room temperature of popc lipid vesicles for various hepes concentrations at ph 4, 7, and 8. for comparison, measurements for the symmetric shorter - chain dlpc in hepes at ph 4 are also included (figure 1d). while the scattering peaks index for a single phase in the case of dlpc, the scattering from popc indicates the coexistence of distinct phases. popc in the presence of hepes presents a pair of scattering peaks that shifts to lower q values (smaller scattering angles) similar to dlpc and another that shifts to larger q values (larger scattering angles) as the hepes concentration is increased. upon close inspection, the peaks shifting to high q values are closely spaced doublet peaks at ph 4 and 7 but not at ph 8. in all of the following discussion, the doublet will be treated as a single phase and reported as an average d - spacing. saxs intensity profiles of popc in various concentrations of hepes solutions at ph 4 (a), ph 7 (b), and ph 8 (c). saxs intensity plots of dlpc in hepes solutions at ph 4 (d). the d - spacing for the two popc phases are plotted versus hepes concentration in figure 2, where the average d - spacing is plotted in the case of doublets. phase i, which swells with the addition of hepes, behaves similarly to dlpc while phase ii appears to dehydrate. this phase coexistence is robust to ph changes below and above the larger pka value of hepes indicating that the mechanism responsible for this phase coexistence is present regardless of the protonation state of the buffer. however, ph values affect both the d - spacing (figure 2b) and the relative intensity of the scattering peaks (figure 2a). for example, the steeper increase of d - spacing for phase i is observed at ph 7, while the lowest d - spacing values for phase ii are observed at ph 8. at ph 7, hepes is predominantly zwitterionic (plus a negatively charged fraction), while at ph 8 it is predominantly negative (plus a zwitterionic fraction). although it is tempting to speculate that the two mlv phases correspond to the two different titrated forms of hepes, it is not immediately clear how such demixing can occur on the length scale of mlvs. however, complete demixing of the two buffer forms might not be required. it is conceivable that the ratio between protonated and unprotonated buffer is different in the two phases and may be sufficient to drive phase separation. a comparison of saxs profiles of popc in 200 mm hepes solutions at ph 4, 7, and 8 (a). d - spacing versus hepes concentration for popc multilayers at ph 4, 7, and 8 denoted by,, and, respectively (b). the difference in d - spacing between popc phase i and phase ii (d - spacing) versus hepes concentration at ph 4 and 8, denoted by and, respectively (c). figure 3a compares scattering profiles of popc in three different buffers at ph 8 : 200 mm hepes, 50 mm tris, and 0.5 mm edta. at ph 8 the amine groups of tris have been deprotonated, while edta is mostly zwitterionic. a phase coexistence is present in all cases, and interestingly, 0.5 mm edta has a comparable effect to that of 200 mm hepes suggesting a correlation between the ionic strength and phase d - spacing (figure 2a). in mixtures, however, the effect of hepes is dominant over that of additives for hepes concentrations higher than 200 mm, as shown in figure 3b, c. comparison of saxs intensity profiles of popc in edta, hepes, and tris at ph 8 (a). comparison of d - spacing of popc multilayers in hepes concentration at ph 8 () versus that in the presence of 0.5 mm edta (b) and 50 mm tris (c) denoted by and respectively. the horizontal dotted lines in panels b and c indicate the d - spacing of popc in pure water. the presence of two (or more) d - spacing values for a mlv sample in equilibrium creates an interesting and challenging theoretical question on intermembrane interactions. it is unlikely that the observed phase coexistence is an artifact of sample preparation or history. in addition, the total number of components in the system increases as buffering agents are included, allowing an expansion in the number of possible phases under the gibbs phase rule. this is demonstrated by the systematic changes in d - spacing in figure 2 and by the robustness to ph values and additives. in addition, sample preparation and equilibration followed tested and established procedures used in previous similar studies of lipids in salt and buffer solutions. the measurements reported here were conducted at room temperature (23 c) and in the presence of excess buffer solution. these conditions are far from the phase boundaries of the popc / water phase diagram so artifacts due to proximity to phase boundaries are unlikely. however, the phase separations induced by these buffers have characteristics similar to those previously observed with multicomponent lipid membrane systems. for a given ph value, the gap in d - spacing values between the two phases increases with hepes concentration as shown in figure 2c. since differences in d - spacings are as high as 15, the observed phenomena can not be due solely to membrane thickness variations but rather due to changes in the interlamellar water space. the interesting behavior observed here is that phase ii appears to dehydrate below the value in pure water, while phase i swells. however, the two phases must be in osmotic equilibrium with one another and this is a peculiar feature that should be accounted for in theoretical models of lipid interaction. such models should include a charge regulation mechanism to account for electrostatic effects as well as account for the screening of vdw attraction and possible changes in the bilayer elasticity. the behavior of phase i can be explained by a reduction in vdw attraction and possibly electrostatic charging. the explanation for the phase ii behavior is not immediately obvious, but one important observation is the qualitatively different d - spacing variations for the two phases. for phase ii, the d - spacing shows a relatively flat region after an initial drop at low buffer concentration, while phase i increases steadily. the simplest mechanism that can explain this behavior is a suppression of the undulation repulsion for phase ii. it has been shown that bilayer undulations can add on the order of 7 - 12 to the d - spacing, which is the range of values observed here. once membrane undulations are suppressed at low buffer concentration, addition of more buffer can not further modify the d - spacing of phase ii reaching a minimum as observed. it is important to note that this minimum is still higher than typical values for popc under osmotic stress. this means that the observed phase is not simply due to dehydration but to a change of membrane interaction parameters. assuming that both phases are lamellar, the free energy of the system should present two or more minima when plotted versus interlamellar spacing. this is an unusual feature that needs to be added to existing models of membrane interactions. the two distinct minima in the free energy profile required by the observed phase coexistence must also have comparable depths. in this respect, it would be interesting to quantify the fraction of lipid in the respective phases. while this could in principle be based on the relative intensity of scattering peaks there are complications in this procedure due to variations in form factors as well as integration artifacts due to the long tails of the scattering peaks produced by bilayer undulation. nevertheless, peak intensities in figure 1 indicate that phase i is dominant for popc / hepes at ph 4, while phase ii is dominant at ph 7 and 8. in this respect, it is also interesting to note that a concentration of 0.5 mm edta (figure 3) is sufficient to replicate the results seen with 200 mm hepes at ph 8, while the effect of 50 mm tris is commensurate to hepes at the same concentration and ph. buffer conductivity was used to further explore this behavior (figure 4), where edta buffers yielded a higher conductivity than those containing tris and hepes at ph 8. this suggests that ionic strength may play an important role in lipid organization and therefore phase separation. conductivity of buffers reported as a function of ph and concentration for hepes (ph 4, 7, and 8 denoted by,, and, respectively), edta ph 8 (), and tris ph 8 (). as mentioned above, the swelling behavior of phase i can be explained by a combination of vdw forces reduction and added electrostatic repulsion. comparison of hepes and hepes with edta data in figure 3b shows that addition of edta reduces the swelling of phase i caused by hepes. this indicates that the electrostatic charging due to hepes and edta must have opposing charges. since edta is highly negative it follows that hepes charges popc membranes positively, a result that is consistent with those obtained with dlpc and mops. hepes, tris, and edta induce a clear phase separation of popc multilayers in excess solution, with an increase of d - spacing in one phase and a reduction in the other. since the two phases must be in osmotic equilibrium with each other and the excess solution, the different d - spacing behavior must be due to distinct mechanisms. the swelling of phase i with added buffer components can be explained by a reduction of vdw attraction forces and added electrostatic repulsion. in contrast, the d - spacing reduction measured for phase ii may be explained by a suppression of bilayer undulation possibly due an increase of bilayer bending rigidity. the experimental results in this letter provide a basis for further developing theoretical models to describe membrane interactions. | recent literature has shown that buffers affect the interaction between lipid bilayers through a mechanism that involves van der waals forces, electrostatics, hydration forces and membrane bending rigidity. this letter shows an additional peculiar effect of buffers on the mixed chain 1-palmitoyl-2-oleoyl - sn - glycero-3-phosphocholine (popc) lipid bilayers, namely phase coexistence similar to what was reported by rappolt. for alkali chlorides. the data presented suggest that one phase appears to dehydrate below the value in pure water, while the other phase swells as the concentration of buffer is increased. however, since the two phases must be in osmotic equilibrium with one another, this behavior challenges theoretical models of lipid interactions. |
these include the presence of at least two of the following : clinical and/or biochemical features of hyperandrogenism, menstrual dysfunction, and the appearance of polycystic ovaries on ultrasound, once other endocrine conditions have been excluded. other criteria that can be used include those from the national institutes of health (nih) and the androgen excess society (aes). pcos has estimated prevalence of over 10% in women of childbearing age. besides being associated with infertility, pcos is also associated with a higher incidence of type 2 diabetes mellitus (t2d), endometrial carcinoma, and cardiovascular disease including stroke and coronary heart disease. the exact etiology of pcos is unknown and probably represents a complex interaction between environmental and genetic factors. obesity in women of childbearing age is associated with anovulation, infertility, pregnancy loss, pregnancy - associated complications such as preeclampsia and gestational diabetes, and postpartum complications including hemorrhage as well as higher rates of infant mortality and congenital defects [3, 4 ]. obesity in patients with pcos is also associated with delayed or failed response to fertility treatments including clomiphene citrate, gonadotropins, and assisted insemination [5, 6 ]. the british fertility society advises that fertility treatment should be deferred until women have a body mass index (bmi) of less than 35 or bmi under 30 if they are below 37 years of age. metformin and nonsurgical weight loss measures have been advocated as first - line management for pcos [8, 9 ]. it has been suggested that even a modest loss of up to 5% of the initial body weight can result in spontaneous ovulation, restoration of menstrual cycle regularity, and pregnancy in obese women with pcos [1012 ]. bariatric surgery is the most durable and effective treatment for morbid obesity and also results in the improvement of the metabolic syndrome. with the safety of the laparoscopic approach and improved understanding of the metabolic changes occurring in bariatric patients postoperatively, morbidly obese women with infertility secondary to pcos have resorted to bariatric surgery. historically, epidemiological studies have suggested that the rapid weight loss in the first year or two after bariatric surgery may increase women 's chance of conception. while the incidence of pcos decreases significantly after surgery, there are very few studies assessing fertility before and after bariatric operations. at present, there is no consensus on the role of such surgery in the management of infertility and whether surgery can also be beneficial in women who have a bmi of under 40 kg / m. in this article, we systematically review the published literature to assess the effects of bariatric surgery on fertility in women with pcos. the prisma statement for reporting systematic reviews and meta - analyses of studies that evaluate health care interventions : explanation and elaboration was utilized as a framework for this systematic review. all manuscripts assessing the quantitative effect of gastric bypass, gastric banding, sleeve gastrectomy, and gastric plication on infertility in females with pcos published between 1 january 1974 and 20 march 2015 were considered eligible for inclusion in this systematic review. search databases, pubmed, embase from 1974 to 20 march 2015, and medline and medline non - indexed items, were searched using the following keywords : polycystic ovary syndrome, infertility, bariatric surgery, gastric bypass, laparoscopic, roux - en - y, gastric band, sleeve gastrectomy, and gastric plication. manuscripts lacking quantitative data, those lacking in relevance to the study question, and those relating to male fertility were excluded. results and data were extracted following analysis and critical review of the results section of original manuscripts. this included sample numbers, age, body mass index, and basic demographics. surgical procedure and technique. this list included roux - en - y gastric bypass (rygb), gastric band (gb), gastric plication (gp), or sleeve gastrectomy (sg), open or laparoscopic. comparisons. outcomes were conception rate, pregnancy, biochemical markers of fertility and pcos, and menstrual regularity. study design. the type of study and level of evidence were recorded. each study was individually assessed for risk of bias giving particular attention to funding sources, limitations of study, and conflicts of interest declared in the discussion section. as the literature primarily is composed of epidemiological studies, the principle summary measure is sample conception rates before and following surgery in addition to / or other biochemical markers of fertility and pcos within the same sample. results of studies have been summarized ; however, quantitative data have not been combined in analysis. the 6 manuscripts that were included in the analysis and their results are summarized in table 1.. demonstrated that, after laparoscopic rygb (75 cm roux limb), weight loss was associated with amelioration of pcos - associated symptoms including resolution of menstrual abnormalities in all patients and resolution of hirsutism in 52% of patients. five patients who were unable to conceive preoperatively were able to conceive without the use of hormones postoperatively although the time interval after surgery is not mentioned in this paper. in a study by jamal., 20 patients with pcos were followed up after rygb (75 cm roux limb and 30 cm biliopancreatic limb) for a mean postoperative follow - up of 46.7 months. preoperatively, 50% of the patients with pcos were infertile, 85% had menstrual dysfunction, and 70% had hirsutism. following surgery - induced weight loss, menstrual irregularities were corrected with return of regular cycles in 82% of patients without the need for hormonal treatment. hirsutism completely resolved in 29% and 77.8% of those with t2d that had complete remission. of the 10 patients who did not conceive before surgery, 4 no longer desired pregnancy with the remaining 6 patients becoming pregnant within 3 years of surgery (5 of whom conceived without any hormonal treatment). series of 5 patients who underwent ivf after bariatric surgery, two women with infertility secondary to pcos and male infertility underwent rygb and gastric banding, respectively. both conceived postoperatively following in vitro fertilization / intracytoplasmic sperm insemination and had uncomplicated deliveries. this paper suggests that although ivf appears to be safe after bariatric surgery, ovarian hyperstimulation may present with features similar to complications after weight loss surgery (especially internal herniation after rygb) and a high index of suspicion is thus required. talebpour reported that gastric plication and gastric bypass appear to have a positive effect on fertility in an abstract presented at ifso 2011. 10 women (71%) out of 14 who were infertile preoperatively became pregnant one year postoperatively. out of 87 premenopausal patients who had irregular menstrual cycles preoperatively, 70 (80%) had regular cycles by the end of the first year postoperatively. this work, only published in abstract form, does not compare the effects of the two operations. in another abstract presented by george and azeez at ifso 2013, 156 female patients between the ages of 20 and 50 underwent laparoscopic sleeve gastrectomy (sg) and were followed up 6-monthly. postoperatively, hirsutism resolved in 77 out of 96 patients (80%) and 54 out of 67 showed resolution of radiological evidence of pcos. 132 patients had menstrual irregularities before surgery but all of these returned to a normal menstrual pattern after sg. four out of the 11 patients who were unsuccessfully treated for infertility preoperatively became pregnant postoperatively. this abstract also showed that urinary stress incontinence resolved or substantially improved in over 40% of patients. unfortunately, this work was only published as an abstract and there is no information regarding the mean follow - up period, the preoperative bmis, and the nature of the study. dixon and o'brien found that 2 infertile patients in their cohort became pregnant after laparoscopic gastric banding. unfortunately, the paper does not make the denominator clear and it is not possible to ascertain how many of the 28 women with primary or secondary infertility were followed up 1 year postoperatively. the definition of infertility, the age of the patients, and the operations were different. controls were not present in the considered papers with patients being control of themselves before and after bariatric surgery. it was difficult to carry out analysis for bias in studies that were only presented as conference abstracts. another bias deals with one of the analyzed studies that considered in vitro fertilization (often performed for the male factor), while the others considered spontaneous pregnancy. women of childbearing age form a significant percentage of patients being referred for and undergoing bariatric surgery. a review of admission data from more than 1,000 us hospitals between 1998 and 2005 revealed that almost half of all patients undergoing inpatient surgical weight loss procedures were women between the ages of 18 and 45. a recent meta - analysis has shown that pcos decreases significantly after bariatric surgery from 45.6% preoperatively to 6.8% at 1 year postoperatively. the reproductive health of female participants was investigated as part of the longitudinal assessment of bariatric surgery (labs-2) study with a self - administered survey within 30 days preoperatively. 42% of women who tried to become pregnant preoperatively in this cohort of patients experienced infertility (defined as 12 months of regular intercourse with a man without contraception but no resulting pregnancy). 65% of these patients however had at least 1 pregnancy after experiencing a period of infertility. a high rate of stillbirths was self - reported by these women with the rate being twice that expected in the usa (13.2 versus 6.2 per 1000 live births). future pregnancies (in the postoperative period) were an important consideration to 30% of patients who were aged 1844 and who did not report natural / surgical menopause, hysterectomy, endometrial ablation, or sterilization (personal or partner). this study revealed that women who were obese by the age of 18 were more likely to report pcos (14.4% versus 5%) and infertility (56% versus 25%) and less likely to have ever been pregnant (75% versus 92%), compared with women whose obesity started after the age of 30. obesity at a young age may be considered an indication for bariatric surgery in effort to prevent infertility developing in later life. the labs-2 study is yet to report the postoperative reproductive health results in this cohort of women. pcos in obese patients primarily manifests itself with irregular or infrequent menstrual bleeding / amenorrhea, hirsutism, and infertility. it is thought that a 5% weight loss can result in resolution of obesity - related anovulation ; however, there is little evidence that this is sufficient in morbidly obese patients. in a retrospective survey, 50% of women (98 patients) aged 40 years or younger with intact uterus and ovaries had anovulatory cycles, defined as cycles of > 35 days longer, prior to bariatric surgery. of these 98 patients, 70 patients (71%) patients who regained ovulation postoperatively had statistically significant greater weight loss compared to those who remained anovulatory. patients who had normal cycles preoperatively still had normal menstrual cycles postoperatively despite the weight loss. in a prospective study of 14 females with pcos, amelioration in clinical symptoms was associated with significant improvements in testosterone, fasting glucose, cholesterol, insulin, and triglyceride levels at 6 and 12 months after rygb when compared to baseline. improvements in biomarkers, hirsutism, and regularity of the menstrual cycles did not correlate with the degree of weight change in this study.. also showed similar improvements in total and free testosterone and amelioration of insulin resistance estimated in a prospective study of 12 premenopausal women with pcos who underwent either rygb or biliopancreatic diversion. traditionally, bariatric surgery has been reserved for patients with a body mass index (bmi) > 40 kg / m or with bmi > 35 kg / m and one or more significant comorbid conditions, when nonsurgical methods of weight loss have failed. recently, the national institute for health and care excellence (nice) in the uk suggested lowering the bmi down to 30 infertility due to anovulation and pcos amongst morbidly obese women could potentially be viewed as an additional indication for bariatric surgery. although most studies show amelioration of pcos postoperatively, to date, the number of studies showing improved fertility is small and they mainly consist of retrospective analysis of small cohorts of patients. as females of childbearing age make up a large percentage of patients undergoing surgery, more research is required in this area of metabolic and bariatric surgery to enable clinicians to advise these women regarding their reproductive health and fertility after surgery. careful follow - up of these patients is required, as pregnancy is usually not advised in the first 1218 months postoperatively. bariatric surgery results in improvement of menstrual irregularities and hirsutism and amelioration of the metabolic profile. however, at this stage, it is difficult to recommend the lowering of bmi criteria for patients with primary infertility and pcos and larger studies are required to confirm the effects of bariatric surgery on fertility and to determine whether different bariatric operations have different results compared to nonsurgical methods of weight loss. polycystic ovary syndrome (pcos) is the most frequent cause of female infertility.pcos, hirsutism, and menstrual irregularities improve after bariatric surgery.the evidence for improvement in fertility after bariatric surgery is still limited.more studies are required to understand which operation (if any) would be best for this cohort of young infertile women. more studies are required to understand which operation (if any) would be best for this cohort of young infertile women. | background. polycystic ovary syndrome (pcos) is the commonest cause of female infertility. visceral obesity and insulin resistance are key pathophysiological mechanisms behind pcos. women suffering from this syndrome and infertility often seek bariatric surgery hoping that they would be able to conceive postoperatively. objective. at present, there is no consensus on the role of bariatric surgery in the management of pcos - associated infertility within the medical community, making it difficult to give specific advice to these women, so a review of the literature was necessary. results. a detailed review of the literature was performed. only 6 manuscripts were relevant and contained quantitative data. they demonstrated that bariatric surgery results in postoperative conception rates varying from 33% to 100%. surgery is also associated with amelioration of menstrual irregularities, hormonal abnormalities, and hirsutism that are associated with pcos. these studies were retrospective and only had a small number of participants with infertility. conclusions. bariatric surgery has been shown to conclusively improve life expectancy, quality of life, and comorbidities like type 2 diabetes and obstructive sleep apnea. however, further research is required to identify whether weight loss surgery results in significant improvement in fertility of women with pcos and to investigate which operation has the best results. |
primary genital porokeratosis is a rare entity but its inclusion in the differential diagnosis of genital lesions is imperative given the known risk of malignant degeneration. a 64-year - old caucasian male with no personal history of skin cancer presented with a 1 year history of an asymptomatic solitary lesion on the glans penis. in the past, patient had been evaluated by urology for an unrelated issue and during that encounter ; urologist was unable to render a diagnosis. upon referral to dermatology, patient presented with a 6 mm, solitary, erythematous annular macule with a circumferential collaret of scale and a central slightly atrophic region. highlighting the whole lesion with a skin marking pen and wiping away with an alcohol pad led to selective accentuation of the collaret of scale. based on examination, provider diagnosed the patient with a solitary porokeratosis and offered to treat with cryotherapy. given the pain involved, patient declined and agreed to a quarterly follow - up. after 12 months, patient returned to the provider with now a 1.0 cm, solitary, erythematous annular macule with a more prominent central erythema and atrophy [figure 1 ]. beside from the increase in size, histology [figure 2a and b ] revealed a focal lymphocytic infiltrate in the papillary dermis with a discrete area of absent granular zone and dyskeratotic cells in the spinous layer underlying a thin tier of compact column of parakeratotic cells ; the cornoid lamella. a 1.0 cm, solitary, erythematous annular macule with a more prominent central erythema and atropy (a) a thin shave biopsy demonstrating two cornoid lamella at far edges. (b) a magnified view revealed a focal lymphocytic infiltrate in the papillary dermis with a discrete area of absent granular zone and dyskeratotic cells in the spinous layer underlying a thin tier of compact column of parakeratotic cells ; the cornoid lamella porokeratosis as an entity has a broad spectrum of presentation ranging from destructive and disfiguring lesions involving the face to involvement of burn scars to dozens of lesions occurring on sun - exposed extremities. clinical variants include porokeratosis of mabelli, linear porokeratosis, disseminated superficial actinic porokeratosis, and punctuate palmoplantar porokeratosis ; but all these share the common feature of cornoid lamella on histologic examination. a less known clinical variant includes porokeratosis ptychotropica (pp) which was first described by lucker., in 1995. in 1999 described a second case where he used a more descriptive term bilateral perianal inflammatory verrucous porokeratosis. features that distinguish pp from genital porokeratosis include multiple cornoid lamellae on histologic examination, a large pruritic verrucous plaque in the perianal region with satellite lesions. although subadjacent dermal amyloid has been described in a number of pp cases, it has also been noted in other cases of porokeratosis and may just represent a feature of chronic irritation. it remains to be seen whether this has any diagnostic value in differentiating pp from other forms of porokeratosis. porokeratosis of mabelli, though the most common variant, is a rather rare occurrence in the genital region. a thorough review of the literature to only include cases that represent primary genital porokeratosis [table 1 ], shows as few as 26 total cases reported in adult males, three cases in adult females, and a single case of linear porokeratosis in an adolescent male. robinson., reported a case of a 39-year - old nulliparous caucasian female with a 30 year history of perianal lesions starting at 8 year of age and spreading to perineum, medial thighs, and plantar surface of feet by age 23. other similar case reports where authors reported lesions other than genital area were also excluded. we opted to exclude this case because of involvement of areas other than perineum, plus based on description of perianal lesions author suspects that this may in fact have represented a case of porokeratosis ptychotropica which both clinically and histologically mimic genital porokeratosis. summary of published data based on review of literature [table 1 ], it is evident that overwhelming majority of patients affected are middle - aged males (90%) with on average over a year long history before presenting to the dermatologist. no clear propensity towards a solitary or multiple lesions was evident based on the data. given the clonal proliferation, dyskeratotic cells, abnormal keratinocyte maturation, and genetic studies demonstrating overexpression of p53 ; porokeratosis is considered a premalignant condition. progression to nonmelanoma skin cancer (especially bowen 's disease and squamous cell carcinoma) is well - documented and estimates range from 6.9 to 30%. risk factors include history of undergoing radiation therapy, chronic lesions, lesions on extremities, older age, and linear variant. based on clinical examination, differential diagnosis in our patient also included annular lichen planus, bowen 's disease, candidiasis, irritant / allergic contact dermatitis, and zoon 's balanitis. treatment options include cryotherapy, electrodessication, photodynamic therapy, co2 laser, topical 5-fluorouracil (5-fu) and excision. kluger., describes a case of genital porokeratosis that disseminated after a 20 year period where the patient reported symptomatic relief after being treated with 3% topical diclofenac. shave removal was curative in our patient and at 6 month follow - up patient was noted to be disease free. this case and review of literature demonstrates the clinical presentation of genital porokeratosis, it highlights different treatment options, and a higher propensity of males being affected by this condition. | we present a case of a 64-year - old uncircumcised male who initially presented to the provider with a 1 year history of a solitary lesion on the glans penis that was clinically diagnosed as porokeratosis of mabelli. a biopsy on a follow - up visit confirmed provider 's clinical suspicion. this article highlights the progression and treatment options for porokeratosis on the male genitalia. |
small mammals from domestic and peridomestic areas were trapped during august december 2010 in 2 high - altitude localities, golgolnaele (1352n, 3943e, elevation 2,700 m) and mahbere silassie (1339n, 3908e, elevation 2,600 m), and in 1 lower - altitude locality, aroresha (1225n, 3933e, elevation 1,600 m), in the tigray region of the ethiopian highlands. kidney samples preserved in rnalater reagent (qiagen, hilden, germany) and stored at 80c were used for total rna extraction by using the nucleospin rna ii kit (macherey - nagel, dren, germany). screening for arenaviruses was performed by using a pan old world arenavirus pcr targeting the large (l) gene (6). screening for hantaviruses was performed by using a nested pcr assay targeting the hantavirus l gene (7). a total of 201 small mammals from 6 species were screened for arenaviruses and hantaviruses (table). among them, 1 ethiopian white - footed mouse (stenocephalemys albipes) from golgolnaele and 2 awash multimammate mice (mastomys awashensis) from aroresha were positive for arenavirus rna ; 10 white - footed mice from the 2 highland localities (6 from golgolnaele, 4 from mahbere silassie) were positive for hantavirus rna. amplicons were purified and sequenced, and nucleotide sequences were aligned on the basis of the amino acid alignment. phylogenetic analyses were performed on the nucleotide sequences by using a maximum - likelihood (ml) approach (8). species identification was confirmed by sequencing the partial mitochondrial cytochrome b gene (11). voucher specimens from representative rodents have been deposited at the evolutionary ecology group, university of antwerp, and are available from the authors on request. after sequencing of the 3 arenavirus - positive samples, 3 distinct arenavirus sequences were obtained, and an ml tree was constructed for these 3 arenavirus sequences and the partial l gene (340 bp) of representatives of old world arenaviruses (figure 1). the 3 sequences cluster with mobala virus (80% bootstrap support), an arenavirus discovered in praomys sp. in the central african republic in 1983 (10). however, the 3 sequences from ethiopia are not monophyletic ; the 2 sequences from multimammate mice cluster together (94% bootstrap support), but the sequence from the white - footed mouse from golgolnaele is basal to the clade (mobala + m. awashensis virus sequences), with the menekre virus, found in hylomyscus sp. in guinea the sequences from multimammate mice on average differ from those of mobala virus and the sequence from the white - footed mouse by the same order of magnitude in terms of amino acids : 5.0 2.1% and 5.9 2.2%, respectively. the average amino acid difference between the sequence from the white - footed mouse and that from mobala virus was 8.1 2.6%. therefore, these arenaviruses seem to be 2 strains of mobala virus carried by 2 rodent species and found in 2 localities 250 km apart from each other and with an altitude difference of 1,100 m. maximum - likelihood tree of old world arenaviruses showing the position of 3 arenaviruses (boldface ; genbank accession nos. jq956481jq956483) found in kidney samples of awash multimammate mice (mastomys awashensis) and ethiopian white - footed mice (stenocephalemys albipes). the tree was constructed on the basis of analysis of partial sequences of the rna polymerase gene ; phylogeny was estimated by using the maximum - likelihood method with the gtr + i + (4 rate categories) substitution model to account for rate heterogeneity across sites as implemented in the phyml program (8). genbank accession numbers of the virus strains : eu136039, gu830849, ay363902, ef179864, gu979511, gu481071, dq868486, gu182412, fj952385, ab586645, gu830863, gu078661, dq328876, ay363904, eu914110, gu182413. after sequencing of the 10 hantavirus - positive samples, 4 distinct hantavirus sequences were obtained, 2 from golgonaele and 2 from mahbere silassie. figure 2 shows the ml tree for these 4 sequences and the partial l gene (347 bp) of representatives of hantaviruses. the tree is not well resolved, and shrew- and mole - associated hantaviruses do not cluster. two rodent - associated clades are supported : the previously known murinae - associated hantaviruses (69% bootstrap support) and the cricetidae - associated hantaviruses (92% bootstrap support, with 1 exception, rockport, soricomorpha - associated hantavirus). although all 4 sequences were found in s. albipes mice, a murinae species endemic to ethiopia, they do not group with the murinae - associated hantaviruses or with hantaviruses found in other african small mammals, such as bats (2,3) or shrews (12,13). the 4 sequences form a unique, divergent clade with the 2 sequences from mahbere silassie basal to the sequences from golgonaele, which cluster together. the average amino acid difference between the sequences from ethiopia and those from murinae - associated hantaviruses was 27.0 4.0%. because the new amino acid sequences are at least 21.0 4.0% divergent from those of other hantaviruses, we conclude that s. albipes mice are carrying a novel hantavirus. we propose the name tigray virus for this virus because it was found in the tigray region of ethiopia. additional genetic characterization, in particular of the small and medium segments, will be conducted to further clarify the evolutionary relationship of this virus within the hantavirus genus. maximum - likelihood tree of hantaviruses showing the position of the 4 sequences of tigray hantavirus (boldface ; genbank accession nos. jq956484jq956487) found in kidney samples of ethiopian white - footed mice (stenocephalemys albipes). the tree was constructed on the basis of analysis of partial sequences of the rna polymerase gene ; phylogeny was estimated by using the maximum - likelihood method with the general time reversible + i + (4 rate categories) substitution model to account for rate heterogeneity across sites as implemented in the phyml program (8). genbank accession numbers of the virus strains : ab620030, nc_003468, eu929078, ef619961, jf276228, ef540771, ef543525, ef397003, nc_005235, ab620033, jn037851, fj170809, fj170812, ab620108, ef641807, fj593501, gq306150, af005729, eu788002, ab620102, fj593498, fj593497, ef646763, nc_005225, gu566021, fj809772, hm015221, aj410618, dq268652, jq082305, eu424336, nc_005238, am998806, nc_005217, dq056292, ef050454, jn116261, eu001330, aj005637, jq287716. two rodent species living in close proximity to human settlements in ethiopia are carriers of arenaviruses and hantaviruses. recently, several new arenaviruses and hantaviruses have been described in small mammals in africa, but no clear association with human diseases has been found (2,3,9,1113). however, arenavirus and hantavirus infections are likely severely underreported because symptoms may resemble those of many other febrile infections (2). investigating the presence of antibodies for mobala virus and the proposed tigray virus in humans in the ethiopian highlands is the next step in evaluating their pathogenicity. a recent study in guinea showed that 2/68 patients with fever of unknown origin had antibodies for sangassou hantavirus (5) ; a case of putative hantavirus disease (hemorrhagic fever with renal syndrome) was also reported in the central african republic (14). hantavirus infections may thus be an unrecognized medical problem in africa and deserve more attention. in conclusion, our screening of 201 small mammals led to the identification of 2 novel strains of mobala arenavirus and a novel hantavirus in 2 rodent species found in ethiopia, m. awashensis and s. albipes. these rodents belong to the exclusively african praomyini tribe (15), which hosts 5/11 arenaviruses (lassa, mopeia, luna, mobala, and menekre viruses) and the only murinae - associated hantavirus (sangassou virus) described in africa. our results support a major role for praomyini as hosts in the evolutionary history of arenaviruses and hantaviruses in africa. | we investigated synanthropic small mammals in the ethiopian highlands as potential reservoirs for human pathogens and found that 2 rodent species, the ethiopian white - footed mouse and awash multimammate mouse, are carriers of novel mobala virus strains. the white - footed mouse also carries a novel hantavirus, the second murinae - associated hantavirus found in africa. |
the kidney plays a key role in maintaining potassium ([k ]) homeostasis by excreting excess potassium. potassium excretion primarily depends on renal (about 90%), and to a lesser extent (about 10%) on colonic excretion1). however, non - renal excretion of [k ] and dialytic [k ] removal are important in regulating potassium balance in esrd patients on hemodialysis because of markedly decreased renal excretion of potassium. total body potassium is approximately 50mmol / kg body weight and 2% of total body potassium is in the extracellular fluid (ecf) compartment and 98% of it in the intracellular fluid (icf) compartment2). oral [k ] intake is initially absorbed in the intestine and enters portal circulation. and then, increased ecf[k ] stimulates insulin release and in turn, insulin facilitates [k ] entry into intracellular compartment by stimulating cell membranena - k atpase3). if it is not for the rapid shift of [k ] from the ecf to icf compartments, serum [k ] increased acutely. excretion of an oral [k ] load in the kidney and colon is a relatively slow process, requiring 6 - 12 hours to be completed. so without rapid transcelluar shift of serum [k ] in the human body, we are exposed to hyperkalemic milieu for a while1). in cases of esrd patient on maintenance hemodialysis, hyperkalemia seems to be primarily related to poor dietary compliance such as too much [k ] intake, inadequate dialysis due to noncompliance or vascular access problems, medications such as aceis, [k ] sparing diuretics, non - selective beta blockers, nsaids, and unfractionate heparin use4). the prevalence of hyperkalemia in any given month of hd patients was reported to be about 8.7 - 10% depending on individual centers5). mortality related to the hyperkalemia has been shown to be about 3.1/1,000 patient - years and mainly related to cardiac rhythm disturbances. so, it is frequently called " a silent and a potential life threatening killer " among patients with esrd under maintenance hemodialysis6). in contrast to hyperkalemia, much less attention has been paid to the hypokalemia in hemodialysis patients because of the low prevalences under maintenance hemodialysis patients. hypokalemia increases some risks of ventricular arrhythmias in patients with underlying cardiac diseases and a higher incidence of ventricular arrhythmias was reported to increase from 9 to 40% during hd in some studies7). recently, the numbers of the new patient undergoing maintenance hemodialysis are tremendously increasing worldwide. the cause of excess mortality in these patients seems to bevarious, but dyskalemia is a common cause among the patients with esrd undergoing hemodialysis. in this article, we are going to review [k ] homeostasis in esrd and how dyskalemia influences morbidity and mortality in maintenance hemodialysis patients. potassium plays various roles in the body maintenance of the resting membrane potential and neuromuscular functioning, intracellular acid - base balances, water balances, maintenance of cell volume, cell growth, dna and protein synthesis, and enzymatic functions8). daily [k ] intake is estimated to range between 50 - 100mmol, of which 90% of [k ] intake is excreted by the kidney and the remainder by the colon. complete excretion of ingested [k ] can be excreted by the kidney in a 6 - 12 hour period1). therefore short - term maintenance of ecf [k ] concentration depends on extra - renal mechanisms that can respond within a minutes. the factors stimulating [k ] shifts from the ecf to icf compartments include insulin release, cathecolamines, metabolic alkalosis, and anabolic state. reverse processes happen in mineral acidosis, hyperosmolarity, non - selective beta - blockade use, and alpha-1 stimulation. potassium is freely filtered at the glomerulus and approximately 65% of filtered load is reabsorbed in the proximal tubule. the collecting duct is the main site of the [k ] secretion into the urine8). factors affecting renal potassium excretion include ; distal nephron sodium delivery, the renin - angiotensin - aldosterone system activation, vasopressin status, dietary potassium intake, acid - base status, distal nephron urine flow, and serum potassium concentration. potassium secretion into the lumen of the distal nephron is passive and this passive movementof potassium is dependent on the concentration gradient across the luminal membrane, the lumen negative electrical gradient (primarily generated by sodium reabsorption) favoring secretion, and the luminal membrane 's permeability to potassium9). aldosterone binds to the nuclear mineralocorticoid receptor within the distal tubule and the principal cells in the cortical collecting duct. at a cellular level, aldosterone opens apical [na ] channels and enhances na - k atpase activity on the baso - lateral membranes, resulting in an increase in [k ] secretion10). the major stimuli for aldosterone secretion are angiotensinii and elevations in serum [k ] level11). aldosterone also influences extra - renal regulation of [k ] secretion via increases in colonic and salivary secretion of [k]12). hyperkalemia is defined as a serum [k ] concentration - greater than 5.0meq / l. the kidneys are primarily responsible for [k ] excretion in healthy adults, it is not surprising that patients with esrd are at a high risk population for developing hyperkalemia. at any given level of kidney function, hyperkalemia is more likely to occur in patients who have concurrent medical conditions such as insulin deficiency or concurrent use of drugs to interfere with [k ] secretions such as aldosterone antagonists, angiotensin ii converting enzyme inhibitors, and/or angiotensin ii receptor antagonists9). patients with chronic renal failure on maintenance hemodialysis develop variety of adaptations to compensate for the decrease of renal [k ] excretion. in this population, extra - renal colonic [k ] excretion is a paramount importance in defending against hyperkalemia. this colonic [k ] adaptation is mediated by increased colonic secretion, which is 2 - 3 fold higher in patients on hemodialysis than in patients with normal renal function13,14). the process of [k ] adaptation is facilitated by the increase in aldosterone secretion associated with increases in serum [k ] concentration. the other system defends against hyperkalemia by regulating distribution of potassium between the intracellular and extracellular compartments. insulin and cathecolamines are major factors to regulate na - k - atpase activities over the short term period15). insulin have a major role in potassium adaptationin end - stage renal disease (esrd), enhancing cellular [k ] uptake16,17). although bicarbonatealone has little or no effect on cellular [k ] uptake inpatients with esrd18), it appears to facilitate insulin'seffect perhaps by correction of acidemia19). clinical data regarding the role ofaldosterone in [k ] handling in dialysis patients is equivocal20). however, with recent controlled trial fails toshow a convincing [k]-lowering effect of fludrocortisones21). it appears to decrease cellular uptake of [k ] via an increase of intracellular calcium, which suppress oxidative metabolism and atp generation of cell and reduce na - k - atpase activity22). extracellular hypertonicity, usually seen with diabetic hyperglycemia23) or hypertonic fluid administration24), causes hyperkalemia on the basisof convective [k ] efflux from cells and insulin deficiency. lastly, severe exercise25) and hemolysis in the course of hemodialysis26) release large amount of [k ] into the ecf compartment and may cause severe hyperkalemia. true hyperkalemias on maintenance hemodialysis occur by increased dietary [k ] intake in interdialytic interval, absence of residual renal function which is the main sources of [k ] removal in the normal persons, reduction in dialysis [k ] clearance, hypoaldosteronism, metabolic acidosis usually seen in esrd, hypercatabolic state, blood transfusions, abnormal colonic [k ] secretion, and various drugs used in hemodialysis patients (e.g. cox 1&2 inhibitors, beta blockers, ace inhibitors, angiotensin receptor blockers, postassium sparing diuretics, succinylcholine, digoxin, cyclosporine, tacrolimus, ketoconazol, potassium containing drugs)3,9,20,24,27). chronic renal failure is the most common cause of [k ] retention, especially when gfr falls toward 20% of normal. in most patients with nonoliguric chronic renal failure, mild hyperkalemia is usual28). in cases of chronic renal failure due to diabetes mellitus and tubulointerstitial diseases, many drugs used in the intensive care unit (icu) can produce hyperkalemia by decreasing [k ] excretion30). shift of the intracellular [k ] to extracellular compartment may lead to severe hyperkalemia in critically ill patients. the traditional concept wasthat metabolic acidosishas been implicated as a causative factor of hyperkalemia31). this paradigm has been disproved, and changes in serum [k ] in relation to acid - base disorders are more complex than initially thought. the most common forms of acute metabolic acidosis in critically ill patients, diabetic ketoacidosis and lactic acidosis, are not associated with shift [k ] out of cells32). hyperkalemia seen with diabetic ketoacidosis is most likely caused by increased release of intracellular [k ] due to the breakdown of muscle cells. hypertonicity of the extracellular fluid causes water to exit cells, and [k ] is swept out along with water. unless renal function is not adequate to eliminate the excess [k ], hyperkalemia occurs. this clinical setting may occur in patients with uncontrolled diabetes and renal insufficiency and can lead to severe hyperkalemia33). massive tissue breakdown such as trauma, burn, and rhabdomyolysis, can lead to release of [k ] into the extracellular space. if renal functions are severely impaired, hyperkalemia may develop. drugs that impair [k ] entry into cells can affect the transmembrane balance of [k ]. -adrenergic blockers inhibit the entry of [k ] into cells and, in cases with renal failure, can accelerate development of hyperkalemia34). succinylcholine blocks normal reentry of [k ] into cells after depolarization and causes a temporary increase in serum [k]35). digoxin, an inhibitor of cell membrane na - k - atpase, impairs [k ] entry into cells, but does not cause significant hyperkalemia at usual therapeutic dose36). however, it sometimes may cause hyperkalemia with toxic doses37). however, it affects cardiac and neuromuscular cells which are particularly sensitive to changes in serum [k ]. in patients with mild to moderate hyperkalemia, patients usually complain of muscle weakness, fatigue, paresthesias, palpitations, and cardiac arrythmias. as evidenced by characteristic changes in the electrocardiogram(ecg) that serve as indicators of potential life - threatening arrhythmias, the first sign of increased serum [k ] is tenting of the t wave. ecg changes include widening of the qrs complex, progressive development of atrioventricular conduction blocks, slow idioventricular rhythm, an ecg tracing that looks like a sine wave, ventricular fibrillation, and finally asystole38). ecg findings are not always sensitive to changes in serum [k ] levels and there is no absolute level of serum [k ] associated with a particular ecg abnormality. sometimes, normal ecg findings have been described with severe hyperkalemia, and in some cases the first manifestation of cardiac changes from hyperkalemia may be ventricular fibrillation39,40). hyperkalemia can cause neuromuscular symptoms such as paresthesias and weakness in the arms and legs, followed by a symmetrical flaccid paralysis of the extremities that ascends toward the trunk, finally involving the respiratory muscles. regardless of the cause, the primary goal of treating hyperkalemia in hd patients is to prevent adverse cardiac arrythmias. therapy to lower serum [k ] should be started immediately when serum [k ] level is greater than 6.5meq / l or if the ecg changes show sings of conduction abnormalities. treatment modalities are aimed at one of three mechanisms to prevent or decrease these complications : (1) direct antagonism of hyperkalemic effect on the cell membrane polarization, (2) movement of extracellular [k ] into the intracellular compartment, and (3) removal of [k ] from the body. however, in patients with hemodialysis, residual renal function is not sufficient to promote kaliuresis.if hyperkalemia is not urgent, sodium polystyrene sulfonate (kayexalate) and calcium polystyrene sulfonate (kalimate) can be used. at equilibrium, each gram of resin removes about 0.5 to 1meq of [k ]. the usual dose of [k]-binding resins are 15 to 30 g orally. when given by mouth, [k]-binding resin has little effect for 4 - 6 hours because it must transit to the colon to be effective. when given as a retention enemas consisting of 30 to 50 g of the resin in 70% sorbitol solution, it works within 1 hour. it is important, however, that the enema should be retained for at least 30 to 60 minutes to obtain the desired therapeutic effect. unfortunately, the combined use of sorbitol and kayexalate can cause bowel necrosis and perforation. these complications seem to be more likely in severely immunocompromised patients or shortly after surgery42). in acute cases when serum [k ] needs to be corrected rapidly, hemodialysis can quickly remove 70 to 150meq of [k ] and should be used asa gold standardtreatment modality when other treatments fail43). in addition to the implementation of rapid treatment, the causes of hyperkalemia should be sought and immediatedlycorrected, and offending drugs should be discontinued when possible. in cases of chronic hyperkalemia, dietary restriction of [k ] is the mainstay of management in these patients (40 - 70meq / day). if acidosis is present, sodium bicarbonate is helpful by increasing distal nephron sodium delivery, inducing kaliuresis, and promoting intracellular potassium shift. an additional alternative is the use of [k]-binding resins such as kayexalate or kalimate combined with sorbitol to avoid constipation, at smaller doses given daily or every other day. hypokalemia usually occurs as a consequence of [k ] depletion due to either increased excretion or inadequate intake. however, shift of [k ] in the extracellular to intracellular compartmentsalso can cause hypokalemia. in cases of esrd patients on hemodialysis, hypokalemia is a relatively rare event comparing to hyperkalemia. the precise prevalence of hypokalemia in maintenance hd patients is unknown but the prevalence is various among different centers5,44,45). most hypokalemic patients are asymptomatic depending on serum [k ] levels but it can be associated with mild muscle weakness to serious sudden cardiac death. the consequences of hypokalemia result to alterations in the resting membrane potential of cardiac and neuromuscular cells. the most serious and potentially fatal effects of hypokalemia are related to disturbances in cardiac rhythm that can lead to cardiac arrest. however, cardiac arrest caused by hypokalemia occurs almost exclusively in patients with underlying cardiac disease or patients taking digitalis. characteristic electrocardiographic (ecg) changes associated with hypokalemia include broad, flat t waves, st depression, the appearance of u waves, qt interval prolongation, and finally ventricular arrhythmias leading to cardiac arrest46). when serum [k ] is less than 3.0meq / l, generalized weakness can develop and serum [k ] decreases to less than 2.5meq / l, muscle necrosis and rhabdomyolysis can occur. with progression of hypokalemia, an ascending muscle paralysis develops, leading to respiratory failure and arrest5,8). hypokalemia in maintenance hemodialysis patients is less frequent condition compared to hyperkalemia (0.3 - 0.5% vs. 8.7 - 10%)5) and can be caused by low dietary potassium intake, malnutrition, chronic diarrhea, prescription of drugs that can increase colonic [k ] excretions such as mineralocorticoids and imprudent use of [k]-exchange resins8,45). one recent study conducted on non dialysis dependent (ndd)-ckd population, overall mortality was significantly associated with both higher and lower serum potassium levels even after adjustments for relevant confounders. they also found that hypokalemias were significantly associated with faster loss of kidney function over time, even after adjusting for other known risk factors such as bp, proteinuria and comorbid conditions. they concluded that hypoand hyper - kalemia are associated with higher mortality in ndd - ckd patients47). the goal of treatment in hypokalemia on maintenance hemodialysis is to prevent cardiac rhythm disturbances and serious neuromuscular weaknesses. supplementation of [k ] is the main treatment for hypokalemia and is usually achieved with the oral administration of potassium preparations. there are four types of potassium preparations : potassium chloride, potassium phosphate, potassium bicarbonate and potassium citrate. conditions requiring emergent therapy are usually rare. in general, plasma [k ] decreases by approximately 0.3meq / l for each 100meq decrease in total body [k ]. potassium replacement should be given orally except when severe hypokalemia is associated with respiratory or cardiac instability, in which case the iv route is prefered. when given intravenously, the rate of [k ] administration should not exceed 20mmol / hourto minimize possible iatrogenic hyperkalemia. for iv infusion of [k ], when potassium is administered intravenously through a peripheral vein, the concentrations should not exceed 50mmol / l. iv fluids containing higher [k ] concentration are often painful and cause peripheral vein irritation. serum [k ] level should be followed closely, especially when using iv route or higher doses, to prevent the development of hyperkalemia because esrd patients with mhd have no residual renal function to excrete excess potassium48). dyskalemia is a frequent electrolyte imbalance observed among the maintenance hemodialysis patients. in case of hyperkalemia, it is frequently " a silent and a potential life threatening electrolyte imbalance " among patients with esrd under maintenance hemodialysis. the prevalence of hyperkalemia in hd patients was reported to be about 8.7 - 10%. mortality related to hyperkalemia has been shown to be about 2 - 5% of deaths among patients with esrd5) and about 24% of patients with hd required emergency hemodialysis due to severe hyperkalemia1). in contrast to the hyperkalemia, hypokalemia in maintenance hemodialysis patients is less frequent condition. the precise prevalence of hypokalemia in maintenance hd patients is unknown but the prevalence is various among different centers5,44,45). most hypokalemic patients are asymptomatic depending on serum [k ] levels but it can be associated with mild muscle weakness to serious sudden cardiac death. it can be caused by low dietary potassium intake, malnutrition, chronic diarrhea, prescription of drugs that can increase colonic [k ] excretions such as mineralocorticoids and [k]-exchange resins8,45). much less attention has been paid to the hypokalemia in hemodialysis patients because of the low prevalence under maintenance hemodialysis patients. however, in cases of severe hypokalemia, we also pay attention to prevent cardiac rhythm disturbances and serious neuromuscular weaknesses. | potassium is abundant in the icf compartment in the body and its excretion primarily depends on renal (about 90%), and to a lesser extent (about 10%) on colonic excretion. total body potassium approximated to 50mmol / kg body weight and 2% of total body potassium is in the ecf compartment and 98% of it in the intracellular compartment.dyskalemia is a frequent electrolyte imbalance observed among the maintenance hemodialysis patients. in case of hyperkalemia, it is frequently " a silent and a potential life threatening electrolyte imbalance " among patients with esrd under maintenance hemodialysis. the prevalence of hyperkalemia in maintenance hd patients was reported to be about 8.7 - 10%. mortality related to the hyperkalemia has been shown to be about 3.1/1,000 patient - years and about 24% of patients with hd required emergency hemodialysis due to severe hyperkalemia. in contrast to the hyperkalemia, much less attention has been paid to the hypokalemia in hemodialysis patients because of the low prevalence under maintenance hemodialysis patients. severe hypokalemia in the hemodialysis patients usually was resulted from low potassium intake (malnutrition), chronic diarrhea, mineralocorticoid use, and imprudent use of k - exchange resins. recently, the numbers of the new patients with advanced chronic kidney disease undergoing maintenance hemodialysis are tremendously increasing worldwide. however, the life expectancy of these patients is still much lower than that of the general population. the causes of excess mortality in these patients seem to various, but dyskalemia is a common cause among the patients with esrd undergoing hemodialysis. |
the risk of complications such as fistula, infection, residual penile curvature in hypospadiass surgery is relatively high even though perfect surgeries are performed ; correction of the complications with secondary surgeries are still challenging especially in patients with scarred, inadequate penile tissue (1). it is reported that when we evaluate all kind of complications the rate is between 1 - 90%. several failed hypospadiass surgeries may result in hypovascular, shortened, scarred penis with penile chordee and leave the patient with minimal residual local skin for penile coverage and urethral reconstruction (2). in case of complicated hypospadias repairs, tissue for resurfacement of penis can be acquired from local tissue or from extragenital areas. chronic tissue expansion was described for delicate tissues and in case of large tissue defects. sasaki has described rapid tissue expansion and called it intraoperative sustained limited expansion which provided less tension and better cosmesis for closing large defects (3, 4). besides, vordermark described tissue expansion for penile reconstruction (5). by combining these methods, we used rapid intraoperative expansion technique by using a foley catheter in a failed hypospadias case who had minimal residual skin secondary to infection. a four - year old boy who was treated primarily a year ago in another center for penoscrotal hypospadias encountered infection following the first surgery which resulted in scarred, inadequate penile skin for reconstruction. the patient still showed penoscrotal hypospadias (figure-1). by combining the advantages of rapid tissue expansion and local tissue usage, we decided to use rapid intraoperative expansion technique using a foley catheter in this failed hypospadias case who had inadequate, fragile, scarred penile skin. informed consent was obtained from parents for publication. under general anesthesia, following appropriate preoperative antibiotics, two incisions of 0.3 cm length were made to dorsum of penis at the corona, fashioned at 11 and 1 o'clock position. then the tips of two 8fr foley catheters were trimmed, keeping the balloons intact. after blunt dissection, the catheters were inserted under the dorsal penile skin (figures 24). balloons were inflated synchronously for four times for three minutes with a two - minute rest period. easy dissection of the skin from the dorsal and lateral sides of the penis was achieved without bleeding. following tissue expansion, adequate penile tissue was achieved to cover the penile shaft through snodgrass repair (figure-5). in four months follow - up, the penis has an optimal esthetic appearance with no fistula and with good urethral calibration (figures 6 and 7). we used rapid intraoperative expansion technique by using foley catheters in a failed hypospadias case who had minimal residual skin secondary to infection. in the literature, foley catheter has been described as atool for rapid expansion in cleft palate repair, alopecia repair, tissue defect repair following mass excision such as basal cell carcinoma, melanocytic nevus from forehead, temple, eyelid, periorbital region and extremities (4, 610). the aim of this paper is to describe the use of foley catheter in cripple hypospadias surgery for rapid intraoperative tissue expansion with easier dissection and less bleeding. tissue for resurfacement of penis can be obtained from local tissue or from extragenital areas such as buccal mucosa and skin. extragenital skin grafts may result in loss of the graft, graft shrinkage, lack of sensation, hair bearing skin with different pigmentation (1, 4). methods for local tissue resurfacement are dorsal relaxing incision, z - plasty, rotation of a scrotal flap and burying the penis into the scrotum. tissue expansion is especially useful in those who lack enough penile skin and extragenital skin graft is not preferred. in the literature, authors reported that tissue expansion provides increased tissue area which is well - vascularized, sensate skin and excellent tissue match for pigmentation, texture and thickness (2, 4, 8, 11). it also avoids the need to transfer tissue over a distance. for these reasons, local tissue expansion has become a good alternative to provide skin for penis (2). another advantage of local tissue expansion is the recruitment of additional skin with a similar distribution of androgen receptors for penile reconstruction as hypothesized by kajbafzadeh. recently, besides chronic tissue expansion, rapid tissue expansion technique has also been used in different parts of the body by some authors. according to auletta immediate tissue expansion has provided enough tissue to close defects up to 5 cm in diameter with 16 - 6% tissue expansion and according to baker and johnson although actual amount of skin increased during expansion is uncertain, it has been estimated as 31% (3, 6, 13). although the authors described expansion of 30%, our case had nearly 10 - 15% of expansion. the reason for this small amount may be the small dimension of the scarred penis. demirseren. described immediate tissue expansion with foley catheter in a case of congenital melanocytic nevus and emphasized the advantage of omnidirectional expansion (7). expanders require long periods of time for expansion, may cause erosion, they are for single - use, expensive and difficult to use in children. meanwhile, foley catheters are readily available, inexpensive, disposable and practical for intraoperative tissue expansion (1, 9, 14, 15). chronic tissue expansion has a potential risk of infection in children as discussed by friedman. however this was n't a significant problem in mathews series (2, 11). also, chronic tissue expansion can be slightly uncomfortable to the patient, may create an unnatural distortion of the dorsum of the penis, requires multiple outpatient clinic visits, repeated inflations and may have psychological implications. according to friedman. the major complications of chronic tissue expansion were implant exposure, deflations, wound dehiscence and minor technical complications with port, hematoma and significant pain (11). the authors describe the method as three to four cycles of inflation and deflation after placement of catheter for 3 to 5 minutes (3). another advantage of rapid expansion is that it does not create a fibrous capsule in the expanded tissue because of brief duration avoiding retraction (4). another advantage of this technique is to provide easier dissection with less bleeding in secondary cases and scarred tissues. by using this method, we were able to both dissect the dorsal and lateral penile skin easily without bleeding and also close the ventral defect. the use of rapid intraoperative tissue expansion in failed hypospadias cases is an effective and feasible reconstructive method which provides additional sensate, hair bearing local tissue with excellent match of texture, pigmentation and thickness to the native penis. | abstractfailed hypospadiass cases may result in hypovascular, scarred penis with residual penile chordee and leave the patient with minimal residual skin for penile resurfacing and urethroplasty. local tissue expansion has become a good alternative to provide skin for penis by using expanders however they require long periods of time for expansion. besides, rapid tissue expansion was also described in different tissues. we used rapid intraoperative expansion technique by using a foley catheter in a failed hypospadias case who had minimal residual skin secondary to infection and we concluded that rapid intraoperative tissue expansion with foley catheter is an effective, feasible reconstructive method for easy dissection and penile resurfacing in failed hypospadiass cases. |
patients who continue to experience seizures after receiving adequate dose of an initial benzodiazepine followed by a second acceptable anti - epileptic drug (aed) is considered refractory. when bolus intermittent therapy or aed fails, continuous infusion of anesthetic agents is recommended. super - refractory status epilepticus is defines as status epilepticus that continues or recurs 24 h or more after the onset of anesthetic therapy, including those cases where status epilepticus recurs on the reduction or withdrawal of anesthesia. around 10 - 15% of patients with various therapeutic options like anesthetic drugs, aeds, magnesium, pyridoxine, steroids, immunotherapy, ketogenic diet, hypothermia, neurosurgery, electrical stimulation therapy and cerebrospinal fluid drainage have been tried with variable success. evidences supporting the management of super - refractory seizures is poor due to lack of randomized or controlled studies and is mainly derived from case reports and retrospective series. ketamine is an n - methyl - d - aspartate (nmda) receptor antagonist, not associated with cardiorespiratory depression. studies have demonstrated efficacy and safety of ketamine for the treatment of refractory status epiepticus (rse). here we report the successful management of two cases of super - refractory status epilepticus in patients with septic shock using ketamine infusion. a 23-year - old lady, around 50 kg was a known case of seizure disorder since 10 years. despite on her regular anti - epileptic medications, she presented with convulsive status epilepticus, which did not respond to intravenous lorazepam bolus and loading dose of phenytoin. dose of her regular medications were maximized (phenytoin, sodium valproate, levetiracetam and phenobarbital). serum phenytoin level was therapeutic and nmda receptor igg antibodies were not detected in serum. she was admitted to intensive care unit (icu) and started on midazolam infusion, which was titrated up to 20 mg / h. patient was intubated and required noradrenaline infusion at 0.15 g / kg / min for the management of hypotension due to septic shock due to urinary tract infection. ketamine 50 mg was administered as a bolus and continued at 100 mg / h. over the next 48 h, convulsive seizure episodes progressively decreased. midazolam and noradrenaline her level of consciousness progressively improved over next 36 h. she was weaned off the ventilator and extubated. a 30-year - old lady (35 kg), known case of chronic kidney disease grade v, on regular maintenance hemodialysis, was admitted to icu following desaturation during hemodialysis. she was intubated and was on mechanical ventilatory support. on 4 day of icu admission, she developed convulsive seizures that did not respond to midazolam bolus and phenytoin loading. phenytoin, sodium valproate, levetiracetam and clobazam were started as maintenance anti - epileptic agents at maximum therapeutic dose. convulsive seizures persisted even after 24 h of midazolam infusion. except for elevated creatinine level (320 mol / l), computed tomography of head and magnetic resonance imaging of brain did not reveal any abnormalities. in the mean time, she developed septic shock, due to acinetobacter ventilator associated pneumonia (vap), requiring noradrenaline infusion up to 0.2 g / kg / min. frequencies of convulsive seizures decreased progressively followed by total absence of convulsive seizures over next 24 h. eeg showed no seizure activities. after the next 24 h free of clinical seizure, ketamine was decreased and stopped. her level of consciousness improved, but her inotropic requirement increased, and the patient expired after 3 days due to multiorgan dysfunction following septic shock. a 23-year - old lady, around 50 kg was a known case of seizure disorder since 10 years. despite on her regular anti - epileptic medications, she presented with convulsive status epilepticus, which did not respond to intravenous lorazepam bolus and loading dose of phenytoin. dose of her regular medications were maximized (phenytoin, sodium valproate, levetiracetam and phenobarbital). serum phenytoin level was therapeutic and nmda receptor igg antibodies were not detected in serum. she was admitted to intensive care unit (icu) and started on midazolam infusion, which was titrated up to 20 mg / h. patient was intubated and required noradrenaline infusion at 0.15 g / kg / min for the management of hypotension due to septic shock due to urinary tract infection. ketamine 50 mg was administered as a bolus and continued at 100 mg / h. over the next 48 h, convulsive seizure episodes progressively decreased. midazolam and noradrenaline her level of consciousness progressively improved over next 36 h. she was weaned off the ventilator and extubated. a 30-year - old lady (35 kg), known case of chronic kidney disease grade v, on regular maintenance hemodialysis, was admitted to icu following desaturation during hemodialysis. she was intubated and was on mechanical ventilatory support. on 4 day of icu admission, she developed convulsive seizures that did not respond to midazolam bolus and phenytoin loading. phenytoin, sodium valproate, levetiracetam and clobazam were started as maintenance anti - epileptic agents at maximum therapeutic dose. convulsive seizures persisted even after 24 h of midazolam infusion. except for elevated creatinine level (320 mol / l), computed tomography of head and magnetic resonance imaging of brain did not reveal any abnormalities. in the mean time, she developed septic shock, due to acinetobacter ventilator associated pneumonia (vap), requiring noradrenaline infusion up to 0.2 g / kg / min. frequencies of convulsive seizures decreased progressively followed by total absence of convulsive seizures over next 24 h. eeg showed no seizure activities. after the next 24 h free of clinical seizure, ketamine was decreased and stopped. her level of consciousness improved, but her inotropic requirement increased, and the patient expired after 3 days due to multiorgan dysfunction following septic shock. a 23-year - old lady, around 50 kg was a known case of seizure disorder since 10 years. despite on her regular anti - epileptic medications, she presented with convulsive status epilepticus, which did not respond to intravenous lorazepam bolus and loading dose of phenytoin. dose of her regular medications were maximized (phenytoin, sodium valproate, levetiracetam and phenobarbital). serum phenytoin level was therapeutic and nmda receptor igg antibodies were not detected in serum. she was admitted to intensive care unit (icu) and started on midazolam infusion, which was titrated up to 20 mg / h. patient was intubated and required noradrenaline infusion at 0.15 g / kg / min for the management of hypotension due to septic shock due to urinary tract infection. ketamine 50 mg was administered as a bolus and continued at 100 mg / h. over the next 48 h, convulsive seizure episodes progressively decreased. midazolam and noradrenaline her level of consciousness progressively improved over next 36 h. she was weaned off the ventilator and extubated. a 30-year - old lady (35 kg), known case of chronic kidney disease grade v, on regular maintenance hemodialysis, was admitted to icu following desaturation during hemodialysis. she was intubated and was on mechanical ventilatory support. on 4 day of icu admission, she developed convulsive seizures that did not respond to midazolam bolus and phenytoin loading. phenytoin, sodium valproate, levetiracetam and clobazam were started as maintenance anti - epileptic agents at maximum therapeutic dose. convulsive seizures persisted even after 24 h of midazolam infusion. except for elevated creatinine level (320 mol / l), other laboratory investigation reports were normal. computed tomography of head and magnetic resonance imaging of brain did not reveal any abnormalities. in the mean time, she developed septic shock, due to acinetobacter ventilator associated pneumonia (vap), requiring noradrenaline infusion up to 0.2 g / kg / min. frequencies of convulsive seizures decreased progressively followed by total absence of convulsive seizures over next 24 h. eeg showed no seizure activities. after the next 24 h free of clinical seizure, ketamine was decreased and stopped. her level of consciousness improved, but her inotropic requirement increased, and the patient expired after 3 days due to multiorgan dysfunction following septic shock. super - refractory status epilepticus is difficult to treat and is associated with significant morbidity and mortality. in patients with rse, the efficacy of g - aminobutyric acid (gaba) ergic agents decline as the duration of seizure increases. gaba receptor trafficking causes failure of inhibitory control, increase in neuronal excitation and persistence of status epilepticus despite the presence of gabaergic agents. as seizure progresses, gabaergic drugs lose their potency and require such high doses that they produce toxic adverse effects. on the other hand, number of nmda receptors increases. no likely response was observed when infused at dose lower than 0.9 mg / kg / h, when introduced at least 8 days after seizure onset, or after the failure of seven or more drugs. in both of our patients, ketamine was initiated within 48 h of onset of refractory seizure at the loading dose of 1 mg / kg followed by infusion at 2 mg / kg / h. ketamine was started when four anti - epileptic medications and midazolam infusion failed to control the seizure. episodes of convulsive seizure progressively decreased over 24 - 48 h after starting ketamine infusion. midazolam was successfully tapered in both cases, and subsequent eeg demonstrated absence of seizure. in both patients, ketamine infusion helped in decreasing the requirement of noradrenaline in both cases. in first case, it was tapered and stopped, but in the second case, it was initially tapered, but later, patient deteriorated due to acinetobacter vap and the dose had to be increased again. as demonstrated in our case, ketamine has been shown to confer hemodynamic stability in patients with shock. but recent retrospective study has shown it to be safe when used at dose of up to 10 mg / kg / h for up to 27 days. ketamine can be a safe, effective, reaidly available and economic therapeutic agent for the management of super - refractory status epilepticus in patients with hemodynamic instability. | refractory and super - refractory status epilepticus is a life - threatening neurological emergency, associated with high morbidity and mortality. treatment should be aimed to stop seizure and to avoid cerebral damage and another morbidity. published data about effectiveness, safety and outcome of various therapies and treatment approaches are sparse and are mainly based on small case series and retrospective data. here we report successful management of two cases of super - refractory status epilepticus refractory to anesthetic therapy with midazolam and complicated by septic shock, managed successfully with ketamine infusion. |
in the real world (outside the laboratories), individuals continuously encounter circumstances and events the consequences of which are, more often than not, uncertain. the presence of uncertainty is an indication that the individual is yet to acquire the specific internal representation that reliably predicts its current environment. in other words, it needs to learn about the stimuli that are associated to and predict the occurrence of important outcomes, so that they can be anticipated with the least amount of uncertainty and consequently, risk. this is best illustrated in the context of predator - prey interactions, where a close match between perception and actual reality is advantageous as it allows individuals to avoid mistakes that could have fatal consequences. in a wide range of animal species, for example, nave prey individuals can learn to recognize a predator by being simultaneously exposed to the cue of an injured conspecific paired with predator odor ; through repetition, a prey increases its certainty associated with correctly labeling a newly learned species as a predator. beyond the realm of predation, a plethora of animal studies across functional domains has shown that associative learning enables animals to forage efficiently while avoiding potentially poisonous food, to navigate their environment, securing territories and reproductive success, highlighting the importance of associative learning in shaping adaptive behavior in a wide range of contexts and species, including humans. plants have been omitted from the conversation because no experimental evidence for their ability to learn by association was available, until now. it is logical then, that the adaptive value of associative learning in vegetal species has never been considered. however in light of the new evidence, it is equally logical to expect that, in plants too, associative learning has a range of ecological purposes from foraging to danger avoidance to social interactions above and below ground. for example, a 2014 study demonstrated that plants are able to detect and distinguish between the sound of feeding caterpillars and those caused by wind or singing insects. like in the animal example provided above, nave plants could learn to recognize the presence of a predator by being exposed to the cue of an injured conspecific (i.e., volatile emission) paired with predator sound (i.e., feeding caterpillar). this herbivore - generated acoustic cue alone could then be used by the plant to mount up their chemical defenses in response to subsequent threats of herbivory. a testable hypothesis could be formulated that acoustic cues by virtue of their rapid transmission in the environment reinforce the effectiveness of other known warning signaling systems relying, for instance, on airborne volatiles. if true, learning to associate sounds produced by feeding insects with the release of volatile emissions by plants under attack could reduce a plant 's perceptual uncertainty and enable a rapid pre - emptive response to looming attacks as and when required. as we all know well, it is more effective to anticipate rather than wait for events to present themselves, especially when involving dangerous interactions. accordingly, selection should favor associative learning mechanisms that enable plants to distinguish whether and when cues are indicative of impending harmful or attractive conditions and thus, allow them to take advantage of new resources and avoid novel threats. the ability to learn through the formation of associations involves the ability to detect, discriminate and categorize cues according to a dynamic internal value system. this is a subjective system of feelings and experiences, motivated by the overall sensory state of the individual in the present and its extension via internal representations of the world experienced in the past ; representations that, as mentioned earlier, play a fundamental role in the decision - making process by providing a reference for the kind of expectations that the individual projects in the future. by demonstrating that plants are able to learn by forming associations, firstly and for the simple reason that feelings account for the integration of behavior and have long been recognized as critical agents of selection, plants too must evaluate their world subjectively and use their own experiences and feelings as functional states that motivate their choices. through careful experimental design, the seedlings in our study were allowed to display a number of behavioral responses to inform us the human observers of their cognitive states. in our second experiment, for example, in which some seedlings were asked to make a decision on their growth direction during the evening hours when light would not normally be available, the young plants informed us of their lack of confidence in the neutral conditioning stimulus (cs) as a reliable predictor for the light a decision they, otherwise, rendered most readily during daylight hours. the expression of the conditioned response (cr) in our study is certainly a good indicator of learning, but the absence of the cr in some experimental groups does not necessarily indicate that learning did not occur. keeping in mind that in a conditioning experiment, what an individual does is not the same of what it knows, is it possible that those seedlings asked to perform outside the daylight hours, chose to opt out of performing the conditioned light - foraging behavior ? because the consequences of performing the cr at a time that is misaligned with the internal circadian signals are uncertain (but likely to be energetically costly), is it possible that the plants opted for what they perceived as (but in actuality, was not) a sure betnamely, their innate positive tropism to light as a solution to the uncertainty problem ? as a matter of fact, we do not know whether this is possible or else, but we now have an experimental framework to find out. secondly, the ability to have experiences and feelings, rather than mere sensations, can be explored as a facet of the ability to learn through the formation of associations. if we agree that associative learning and internal value systems based on feelings are evolutionarily linked and constitute what we may refer to as (basic) consciousness, then plants could open a new interface into exploring the processes that have led to the emergence of consciousness (assuming it to be a process that actually emerged or a trait that was acquired through an evolutionary event). by uprooting it from the idea that it is a process or trait that occurs as the intrinsic operation of neural circuitry and thus, it is generated by neurological substrates (as mentioned in the 2012 cambridge declaration on animal consciousness), plants help us to unnerve our premise that consciousness entails attributes derived from specific physical structures such as brains and neurons. moreover, they encourage us to put forward a quantitative theory of consciousness that accounts, more adequately, for its expression through the incredible diversity of living species. just what kind of theory this may be is an open question (but see integrated information theory), but at the very least it should include analyses of behavior and ecophysiology in a wider range of species that transcends the animal kingdom (including the human). and lastly, questions about the cognitive capacities of animals and specifically, animal consciousness often play a role in discussions about animal welfare and moral status. this debate has been recently extended to include plants and as experimental evidence for the cognitive capacities of plants accrues, the controversial (or even taboo) topic regarding their welfare and moral standing and our ethical responsibility toward them can no longer be ignored. | abstractacross all species, individuals thrive in complex ecological systems, which they rarely have complete knowledge of. to cope with this uncertainty and still make good choices while avoiding costly errors, organisms have developed the ability to exploit key features associated with their environment. that through experience, humans and other animals are quick at learning to associate specific cues with particular places, events and circumstances has long been known ; the idea that plants are also capable of learning by association had never been proven until now. here i comment on the recent paper that experimentally demonstrated associative learning in plants, thus qualifying them as proper subjects of cognitive research. additionally, i make the point that the current fundamental premise in cognitive science that we must understanding the precise neural underpinning of a given cognitive feature in order to understand the evolution of cognition and behavior needs to be reimagined. |
cervical spondylotic myelopathy (csm) results in neurologic impairment secondary to degeneration of the structures in the cervical region of the spinal column causing narrowing of the spinal canal. csm is the most common cause of spinal cord dysfunction worldwide.1 traumatic spinal cord injury (sci) leads to varying degrees of motor and/or sensory deficits and paralysis.2 the annual incidence of acute and chronic sci in the united states is more than 10,000, resulting in 720 cases per million persons enduring permanent disability each year. the economic impact of sci is estimated to be more than $ 4 billion annually.3 although csm and sci are the major causes of spinal cord compression, other causes include neoplasm and infection.4 5 anterior spinal cord compressive lesions of the cervical spine are ideally addressed via an anterior approach. the procedure involves the removal of the vertebral body (corpectomy) along with the intervening disk, placement of a graft into the resulting gap, and fixation using a locking anterior cervical plate. anterior cervical corpectomy is the most direct and thorough surgical approach for anterior decompression when the spinal cord compression is located directly behind a vertebral body. indications for this procedure include degenerative disorders,6 neoplasm,4 trauma,7 infectious diseases,5 or correction of degenerative or iatrogenic kyphosis.8 the procedure has demonstrated good clinical results in terms of neurologic outcome and success in arthrodesis and stabilization.9 most reports in the literature indicate an increase in the nonunion rate when fusion is attempted at more than one level, and occasionally this failure of fusion is associated with neurologic aggravation.10 11 although a long iliac bone graft should provide a satisfactory opportunity for fusion, its inherent strength is not sufficient to resist graft fracture and segmental collapse. therefore, a fibular graft is used to circumvent graft failure due to compressive loading across multiple segments. however, the fibular graft needs over a year to incorporate, which may require long - term postoperative immobilization even with halo orthoses.12 as cell viability is maintained in the vascularized bone graft, primary bone healing occurs at the fibula strut vertebral body interface in contrast to creeping substitution into the scaffold of necrotic nonvascularized bone graft. therefore, extensive bone remodeling, including revascularization, resorption, and production of new bone, does not occur as in nonvascularized bone grafts and so structural viability and strength need to be maintained throughout the healing process in the vascularized bone grafts.13 14 also, the resorption of the nonvascularized bone graft during this process leaves the bone so weak that the graft may fracture even after being incorporated.15 we hypothesized that the free vascularized fibular graft (fvfg) could resolve the mechanical and biological problems associated with nonvascularized grafts. to our knowledge, an fvfg has only been used in anterior cervical reconstruction after corpectomy in a few cases. the purpose of our study was to evaluate the clinical and radiographic results of using an fvfg in the anterior reconstruction of the cervical spine following varying levels of corpectomy. following approval of the local institutional review board, 10 patients underwent anterior cervical reconstruction using an fvfg after corpectomy between january 2008 and april 2010. the patients comprised 7 men and 3 women with a mean age of 56 years (range, 45 to 65 years) at the time of surgery. the following inclusion criteria were applied : (1) clinical evidence of csm, (2) motor and/or sensory deficits determined by clinical examination and confirmed by neurophysiologic studies, and (3) minimum of two levels of cervical disk affection documented by magnetic resonance imaging (mri). the mean duration from the onset of symptoms to the time of clinical evaluation was 6 months, ranging from 4 to 12 months. two patients walk with someone else 's help or with the aid of a frame, and 1 patient with gait deterioration reported multiple episodes of falls ; the remaining 3 patients were chair - bound. anteroposterior and lateral radiographs of the cervical spine were obtained and the lordosis angle was measured. mri revealed t2-weighted signal abnormalities within the spinal cord parenchyma in all patients. to standardize each patient 's neurologic assessment, the japanese orthopaedic association (joa), the mean preoperative joa, modified joa, and nurick scores were 9, 9.7, and 3.7, respectively. we used the smith - robinson anterior cervical approach for corpectomy (fig. 1) and a lateral approach to harvest the fibular graft.11 the skin was incised vertically to facilitate the dissection of the recipient vessels and graft placement. after incising the skin and platysma muscle, the dissection was continued carefully to prepare a superficial recipient vein (branch of the external jugular veins). superficial veins are better than deep venae comitantes because they have larger diameters, thicker walls, and better drainage. furthermore, anastomosis is much easier because it is carried superficially in a wide field. based on the levels affected, either the superior thyroid artery or lingual artery was dissected gently and prepared as a recipient vessel. we performed diskectomy followed by corpectomy at the desired levels until removal of the posterior longitudinal ligament, exposure of the dura, and complete decompression of the spinal cord were achieved. intraoperative view of the corpectomy defect with the black arrow pointing to the site of division of the posterior longitudinal ligament. we used the superior thyroid artery in eight patients and the lingual artery in two as the recipient artery, and the venae comitantes or a branch of the external jugular veins as the recipient vein. the length of the free fibular graft was accurately measured according to the size of the cervical defect (fig. we cut the fibula using an electric reciprocating saw while protecting the vascular pedicle at all times. the fibular graft was positioned with the vascular pedicle ending in the cephalic direction and directed to the side of the incision (right or left). the most lateral parts of the vertebral body were not removed during corpectomy to protect the vertebral artery. at the site of exit of the vascular pedicle, the most lateral part of the vertebral body was completely removed to provide passage for the vascular pedicle without compression. harvested free fibular graft after being shaped to match the defect size. a locked anterior cervical plate was applied prior to performing vascular anastomoses to protect the vascular pedicle. then, end - to - end arterial and venous anastomoses were performed using an operation microscope. postoperatively, the neck was immobilized in a rigid collar with chin support all day for 2 weeks and then during the daytime only for another 4 weeks. all patients were clinically evaluated at discharge, monthly for 6 months, at the end of the first year, and annually thereafter according to the clinical evaluation score that was based on three factors16 : (1) neck pain : no pain = 0, mild pain and the patient generally able to perform daily activities = 1, moderate pain that is tolerable with some limitations on daily activities = 2, severe pain with serious limitations on daily activities = 3 ; (2) pain medication : none = 0, non - narcotic medication = 1, and narcotic medication = 2 ; (3) ability to return to work : full return = 0, modified work duties = 1, unable to return to work = 2. the total score (0 to 7) was interpreted as follow : 0 to 1 = excellent (satisfactory), 2 to 3 = good (satisfactory), 4 to 5 = fair (unsatisfactory), 6 to 7 = poor (unsatisfactory). neurologic evaluation was done according to the joa, modified joa, and nurick scores at the same clinical follow - up visits. the neurologic recovery rate was determined using the formula described by hirabayashi,17 in which neurologic recovery rate = [(postoperative joa score preoperative joa score)/(full score preoperative joa score) ] 100 (%). surgical outcome was further quantified by categorizing the recovery rate into five groups previously designated by matsuda : excellent, 75 to 100% ; good, 50 to 74% ; fair, 25 to 49% ; unchanged, 0 to 24% ; and worse, 3.176 degrees) was 0%, and no patient developed cervical kyphosis.22 abbreviation : fsh, fused segment height. subsidence was determined as the change in fsh, with a change of 3 mm defined as significant subsidence.20 in our study, the mean change in fsh was < 1 mm (0.982 mm ; range, 0.23 to 1.65 mm). three patients showed excellent clinical outcome, and the remaining 7 had good clinical outcome. the mean postoperative joa, modified joa, and nurick scores were 14.5, 15.5, and 1.7, respectively. statistical analysis of the differences between the preoperative and postoperative joa, modified joa, and nurick scores was performed using wilcoxon signed rank test and was found to be statistically significant (p = 0.004, 0.005, and 0.007, respectively). the neurologic recovery rate was excellent in 4 patients, good in 5, and fair in 1 (table 3). early complications included serous discharge from the cervical wound in 1 patient, which closed after 2 weeks of antibiotic administration. late complications included pullout of the locked plate 's lower screws, despite successful fusion, in 1 patient at 44 months and adjacent segment degeneration of the c5c6 in another patient at 36 months. delayed wound healing with superficial wound infection was observed in one patient, which was controlled with antibiotic therapy. the advantages of vascularized over nonvascularized bone grafts include rapid consolidation, resistance to infection, hypertrophic reaction guided by mechanical load, and tolerance of therapeutic levels of radiation.23 when successfully revascularized, free vascularized bone grafts maintain their cellular viability as a result of the preserved endosteal and periosteal vascular blood flow. therefore, revascularized bone grafts actively participate in bony union and provide fast and reliable periosteal growth and healing, comparable with fracture healing under the normal physiologic state.24 in most cases of anterior cervical reconstruction, substantial volume and/or length of bone are needed, and high compression strength is desired. the fibula is superior in axial strength ; is easily accessible in the prone, supine, and lateral positions ; it has low donor site morbidity ; and it can be easily harvested simultaneously with spinal surgery.25 two main problems are encountered after strut graft reconstruction following cervical corpectomy : pseudarthrosis and graft complications such as dislocation or fatigue fracture. the literature shows that the use of autogenous nonvascularized fibular grafts or fibular allografts is associated with significant pseudarthrosis or graft displacement when used for anterior cervical reconstruction following corpectomy. fernyhough performed a retrospective study of 126 multilevel corpectomy cases for spondylosis to evaluate the fusion rates using autograft and allograft fibula strut graft for reconstruction.26 the nonunion rate was high in both groups (27% of the autograft group and 41% of the allograft group) and was found to be correlated with increasing numbers of motion segments fused. whitecloud and larocca reported 26 patients with fibula strut graft reconstruction of the cervical spine.12 in the 26 cases, there were 3 graft dislocations and 2 graft fractures. since then, other reports have documented dislocation / fracture rates for bone struts of 0,27 28 2.4,26 3.8,29 6.5,30 6.9,31 7.1,32 7.8,33, 8.7,16 9.1,34 10.2,35 10.8,36 11.1,37 13.6,38 15.0,39 21.4,40 26.3,12 30.0,41 50.0,42 and 71.4%.43 pseudarthrosis, which means failure to achieve successful fusion at the strut graft vertebral body interface with the development of a false joint, remains a serious complication with bony struts, particularly those extending over three or more disk levels, and contributes to recurrent pain with neurologic deterioration and patient disability. it might be associated with hardware failure, graft displacement, or migration that can lead to spinal cord compression.33 pseudarthrosis increases in the presence of several risk factors, including smoking,44 increased number of levels fused, and medical comorbidities such as advanced age, obesity, osteoporosis, chronic steroid use, malnutrition, and chronic illnesses. the choice of the graft material, surgical technique, and use of instrumentation affect the rate of successful fusion.45 it is quite difficult to determine pseudarthrosis rates from the literature because allografted and autografted cases are often mixed together or because autogenous bone grafts obtained by corpectomy are used in conjunction with fibular strut allograft in the same patients.16 where possible to gather data about the fusion for strut grafts, pseudarthrosis rates for autografts (two - level corpectomy) were 12,35 14.3,40 14.8,38 21.4,32 24,46 27,27 and 30%.41 the rates for allografts were 13.4,47 21.21,37 29,46 41,26 and 41%,27 and the pseudarthrosis rate for mixed series of allografts and autografts was 13.0%.16 our study showed solid fusion in all patients in a relatively short time (mean, 3.5 months) compared with a nonvascularized fibular graft, which takes longer to incorporate.12 this short fusion time directly prevented certain complications, such as graft dislocation, dislodgment, and fracture. furthermore, the rapid incorporation of the fvfg and solid fusion resulted in maintenance of lordotic correction by the strut graft, demonstrated by an insignificant loss of lordosis (mean, 0.95 degrees) and fsh (mean, 0.982 mm) and the fact that no patient developed cervical kyphosis during the follow - up. accordingly, one might consider using the vascularized fibular graft reconstruction particularly in cases of corpectomy extending over an increased number of disk levels with an elevated risk of pseudarthrosis. also, due to its resistance to infection, it would be advantageous to replace the destroyed vertebral body in cases of either pyogenic or tuberculous cervical spondylodiskitis. one may consider it after tumor resection due to its tolerance to therapeutic levels of radiation. it also might also be considered in patients with associated comorbidities such as diabetes, osteoporosis, thyroid dysfunction, and rheumatoid arthritis and in patients treated with oral steroids who are more likely to develop pseudarthrosis. we believe that the achievement of rapid and reliable fusion was also reflected in the neurologic recovery rate, which was excellent in 4 patients, good in 5, and fair in 1. we think that the lower screw pullout that occurred in 1 patient might be due to the short period of immobilization, and the adjacent segment degeneration that developed at the caudal disk space of the fused c3c5 in the third month in 1 patient might be related to the short distance between the tip of the plate and the adjacent disk (< 5 mm) and the slight increase in lordotic curvature of the plate. there have been reports of successful multilevel anterior cervical fusion using an fvfg.48 although the number of patients in our study was small, to the best of our knowledge it represents the largest series of patients treated using fvfgs in anterior cervical reconstruction that included both degenerative and infection cases. the use of anterior instrumentation is intended to help prevent graft dislodgment, promote union, and maintain sagittal alignment. routine angiographic evaluation of the vasculature in every patient undergoing free fibula flap surgery is probably unnecessary and expensive and does not provide benefits that outweigh the risks.49 therefore, no preoperative angiographic imaging was obtained in any of the patients in this study. moreover, the use of fvfg in anterior cervical reconstruction involves a complex spinal surgery, with wide surgical exposure and long procedures with extensive blood loss. thus, these patients are susceptible to hypothermia and poor peripheral circulation, with an increased risk of deep venous thrombosis.21 50 however, these complications did not occur in our study. our findings show that use of an fvfg can achieve reliable and successful fusion with both clinical and neurologic improvement ; therefore, we believe it is a valuable and effective technique in anterior cervical reconstruction. | study design prospective study. objective the aim of this study was to evaluate the clinical and radiologic results of using free vascularized fibular graft (fvfg) for anterior reconstruction of the cervical spine following with varying levels of corpectomy. methods ten patients underwent anterior cervical reconstruction using an fvfg after cervical corpectomy augmented with internal instrumentation. all patients were evaluated neurologically according to the japanese orthopaedic association (joa) and modified joa scoring systems and the nurick grading system. the neurologic recovery rate was determined, and the clinical outcome was assessed based on three factors : neck pain, dependence on pain medication, and ability to return to work. the fusion status and maintenance of lordotic correction by the strut graft were determined by measuring the lordosis angle and fused segment height (fsh). results all patients achieved successful fusion. the mean follow - up period was 35.2 months (range, 28 to 44 months). graft union occurred at a mean of 3.5 months. the mean loss of lordotic correction was 0.95 degrees, and the mean change in fsh was < 1 mm. the neurologic recovery rate was excellent in four patients, good in five, and fair in one. all patients achieved satisfactory clinical outcome. no neurologic injuries occurred during the operations. conclusion the use of fvfg is a valuable and effective technique in anterior cervical reconstruction for complex disorders. |
there is an association between body mass index (bmi) and increased early mortality in the general population.1,2 however, while in some studies, both low and high bmi have been associated with increased mortality (i.e., mortality plotted as a function of bmi has a u - shaped curve),1,2 other studies have shown an association between high bmi and increased mortality (i.e., mortality plotted as a function of bmi has a j - shaped curve).1,2 in this sense, subjects with low bmi present with a higher risk of death due to cerebrovascular disease, pneumonia, and diseases of the central nervous system.1,2 on the other hand, increased fat mass (fm) is associated with the development of several chronic diseases, particularly those of the cardiovascular system.1,2 patients with chronic obstructive pulmonary disease (copd) generally present with difficulty in breathing,3 inadequate dietary intake due to difficulties in ingesting food4 and excessive apoptosis of skeletal muscle due to increased systemic inflammation.5 these and other factors lead to a negative energy balance and decreasing body weight over the course of the disease, with the reduction in body weight occurring mainly as a result of a decrease in skeletal muscle mass.6,7 it has been shown that the bmi is an independent predictor of mortality in copd patients.6,7 nonetheless, the association between bmi and mortality seems to differ according to the severity of copd. patients with mild copd present a bi - modal association between bmi and mortality (i.e., mortality plotted as a function of bmi has a u - shaped curve), with the lowest risk of death occurring in normal weight to overweight patients. among patients suffering from severe copd, mortality rate decreases with increasing bmi (i.e., overweight and obese patients have lower mortality rate).810 the observed lower risk of death in overweight and obese patients with copd has been called the obesity paradox;11 although its mechanism is still unknown, the obesity paradox seems to be partly related to the presence of higher fat - free mass (ffm) in these patients.6 exercise intolerance is a major symptom of patients with copd.12 its cause has been shown to be multifactorial and to depend on impairments in airway flow, pulmonary mechanics, metabolic pathways, gas exchange, cardiac performance, respiratory and peripheral muscles and other factors.13 in this context, various studies have investigated the influence of nutritional status on exercise capacity ; it is generally agreed that patients with a low bmi, which is most often due to low ffm, show lower exercise capacity on submaximal and maximal exercise tests.8,1420 however, most of these studies investigated severe copd patients who were underweight or of normal weight.15,16,19 some studies have included overweight or obese patients, but they did not analyze these patients separately.8,14,18,21 the few previous studies that present data on the influence of being overweight or obese on the exercise capacity of patients with severe copd report conflicting results. for example, esnner.20 showed that obese men and women presented lower exercise capacity than normal weight counterparts independent of age, height, fev1/fvc ratio, race, education and smoking. however, these authors included patients with mild to severe copd and did not use the degree of airway obstruction [forced expiratory volume at 1 s (fev1) ] in the adjusted models. ischaki.17 showed a positive association between bmi and exercise capacity in patients with mild to severe copd ; however, their results show no clear association between nutritional status and exercise capacity in severe copd patients who are overweight or obese. thus, the influence of nutritional status on the exercise capacity of overweight or obese copd patients is still unknown. our aim was to investigate the impact of nutritional status on body composition, exercise capacity and respiratory muscle strength in patients with severe copd. we hypothesized that patients who were overweight or obese would have a greater ffm than patients who were of normal weight or underweight. considering that the major contributor to ffm is skeletal muscle mass, we hypothesized that a greater ffm would lead to greater exercise capacity, in part due to greater respiratory muscle strength. thirty - two patients (nine women) with severe copd participated in this prospective cross - sectional study. the patients were enrolled at the pulmonary outpatient clinic of the university hospital of the londrina state university. the diagnosis of copd was based on past smoking history, clinical evaluation and pulmonary function tests showing irreversible airflow obstruction.12 all patients with copd were in a stable state at study entry (no exacerbations in the last three months that required a change in medication or hospital admission), were not currently smoking and were undergoing standard pharmacological treatment for copd.12 none of the patients were undergoing long - term oxygen therapy. the classification of copd severity followed the gold guidelines,12 i.e., severe copd represented fev1 < 50% of predicted value. patients with orthopedic, neurologic or unstable cardiac diseases that might interfere with the results were excluded. the patients were divided into three groups according to the world health organization stratification of bmi:22 overweight / obese (bmi between 25 and 29.9 kg / m, n = 6 ; bmi between 30 and 34.9 kg/ m, n = 2), normal weight (bmi between 18.5 and 24.9 kg/ m, n = 17) and underweight (bmi < 18.5 kg / m, n = 7). overweight and obese patients were analyzed together due to the small number of obese patients in our sample. the ethics committee of the north paran university hospital approved the research protocol and a written consent form was obtained for each patient. spirometry was performed using a turbine portable spirometer (pony, cosmed, rome, italy) according to american thoracic society guidelines.23 the fev1, forced vital capacity (fvc) and maximal voluntary ventilation (mvv) were obtained and expressed as percentage of predicted according to knudson.24 body weight was measured on a calibrated balance to the nearest 0.1 kg while patients were barefoot and wearing light clothing (filizola model 31, filizola, so paulo, sp, brazil). body height was measured to the nearest 0.5 cm while patients were barefoot and standing with their backs and heels touching a vertical bar (filizola model 31, filizola, so paulo, sp, brazil). body composition was assessed by single - frequency bioelectrical impedance analysis (biodynamics 310, biodynamics corporation, seattle, wa, usa). all body composition measurements were performed between noon and 2 p.m. the patients fasted on the day that the measurement was performed and did not ingest coffee, tea, chocolate or alcoholic beverages for 48 h prior to the measurement. during the measurement, patients lay in a supine position with their limbs slightly apart from their bodies. two electrodes were positioned on the dorsal surface of the right hand, and two additional electrodes were positioned on the dorsal surface of the right foot. the ffm was calculated using an equation that has been specifically validated for patients with respiratory diseases.25 fm was calculated as total body weight minus ffm. ffm index (ffmi) and fm index (fmi) were calculated by dividing the body weight (in kg) of ffm and fm, respectively, by height (in m) squared in order to adjust for body surface area.26 it is important to note that the known prerequisites for the use of bioelectrical impedance analysis, which include patient preparation prior to the measurement, adaptation of equations used to transform the measured resistance into ffm or total body volume and testing of the validity of the use of these equations in the populations for which they are intended, were followed in the present study. under these conditions, bioeletrical impedance analysis measurements are considered accurate and comparable to other techniques used to assess body composition.25 the exercise capacity was assessed by the six - minute walking distance test (6mwd), which is validated27 and reliable28 for evaluation of the exercise capacity of patients with copd. patients were instructed to walk as fast as possible, aiming to complete the longest possible distance in the allotted time. at each full minute during the test, the patients were verbally encouraged with a standardized incentive phrase. each patient s result was expressed as an absolute value (meters) and as a percentage of the normal values.29 the strength of the respiratory muscles was evaluated by measuring the maximal inspiratory pressure (pimax) and maximal expiratory pressure (pemax) using an analog manovacuometer built by record (so paulo, sp, brazil). the manovacuometer was connected to an inextensible rubber hose of 38 cm in length and 0.5 cm in diameter ; the hose was connected to a mouthpiece with a 2-mm hole to avoid any influence of intra - oral pressure on the measurements. a nose clip was used to avoid air leakage through the nostrils. for the pimax measurement, the patients began at the residual volume level and performed a maximal inspiration until total lung capacity was reached. for the pemax measurement, the patients began at total lung capacity and performed a maximal expiration until only residual volume remained. maximal pressures were expected to be sustained for at least one second. for each measurement, expected values for pimax and pemax were calculated according to black and hyatt.30 data distribution was verified by the shapiro - wilk test. only fev1/fvc (%) and fmi presented non - parametric distributions. the groups were compared by one - way anova followed by the sheff post hoc, or, when necessary, by the kruskal - wallis test followed by multiple comparisons (z values). these f to enter and remove values were chosen in order to detect variables that were significant at p < 0.05. the walking distance, expressed as a percentage value, was considered as the dependent variable, and only the variables that were significantly associated with the walking distance in pearson s correlations were used in the multiple regression analyses. the results are presented as mean standard error of the mean (sem). a p - value lower than 0.05 was considered significant in two - tailed analyses. all analyses were performed using statistica version 7 software (statsoft, tulsa, ok, usa). thirty - two patients (nine women) with severe copd participated in this prospective cross - sectional study. the patients were enrolled at the pulmonary outpatient clinic of the university hospital of the londrina state university. the diagnosis of copd was based on past smoking history, clinical evaluation and pulmonary function tests showing irreversible airflow obstruction.12 all patients with copd were in a stable state at study entry (no exacerbations in the last three months that required a change in medication or hospital admission), were not currently smoking and were undergoing standard pharmacological treatment for copd.12 none of the patients were undergoing long - term oxygen therapy. the classification of copd severity followed the gold guidelines,12 i.e., severe copd represented fev1 < 50% of predicted value. patients with orthopedic, neurologic or unstable cardiac diseases that might interfere with the results were excluded. the patients were divided into three groups according to the world health organization stratification of bmi:22 overweight / obese (bmi between 25 and 29.9 kg / m, n = 6 ; bmi between 30 and 34.9 kg/ m, n = 2), normal weight (bmi between 18.5 and 24.9 kg/ m, n = 17) and underweight (bmi < 18.5 kg / m, n = 7). overweight and obese patients were analyzed together due to the small number of obese patients in our sample. the ethics committee of the north paran university hospital approved the research protocol and a written consent form was obtained for each patient. spirometry was performed using a turbine portable spirometer (pony, cosmed, rome, italy) according to american thoracic society guidelines.23 the fev1, forced vital capacity (fvc) and maximal voluntary ventilation (mvv) were obtained and expressed as percentage of predicted according to knudson.24 body weight was measured on a calibrated balance to the nearest 0.1 kg while patients were barefoot and wearing light clothing (filizola model 31, filizola, so paulo, sp, brazil). body height was measured to the nearest 0.5 cm while patients were barefoot and standing with their backs and heels touching a vertical bar (filizola model 31, filizola, so paulo, sp, brazil). body composition was assessed by single - frequency bioelectrical impedance analysis (biodynamics 310, biodynamics corporation, seattle, wa, usa). all body composition measurements were performed between noon and 2 p.m. the patients fasted on the day that the measurement was performed and did not ingest coffee, tea, chocolate or alcoholic beverages for 48 h prior to the measurement. during the measurement, patients lay in a supine position with their limbs slightly apart from their bodies. two electrodes were positioned on the dorsal surface of the right hand, and two additional electrodes were positioned on the dorsal surface of the right foot. the ffm was calculated using an equation that has been specifically validated for patients with respiratory diseases.25 fm was calculated as total body weight minus ffm. ffm index (ffmi) and fm index (fmi) were calculated by dividing the body weight (in kg) of ffm and fm, respectively, by height (in m) squared in order to adjust for body surface area.26 it is important to note that the known prerequisites for the use of bioelectrical impedance analysis, which include patient preparation prior to the measurement, adaptation of equations used to transform the measured resistance into ffm or total body volume and testing of the validity of the use of these equations in the populations for which they are intended, were followed in the present study. under these conditions, bioeletrical impedance analysis measurements are considered accurate and comparable to other techniques used to assess body composition.25 the exercise capacity was assessed by the six - minute walking distance test (6mwd), which is validated27 and reliable28 for evaluation of the exercise capacity of patients with copd. patients were instructed to walk as fast as possible, aiming to complete the longest possible distance in the allotted time. at each full minute during the test, the patients were verbally encouraged with a standardized incentive phrase. each patient s result was expressed as an absolute value (meters) and as a percentage of the normal values.29 the strength of the respiratory muscles was evaluated by measuring the maximal inspiratory pressure (pimax) and maximal expiratory pressure (pemax) using an analog manovacuometer built by record (so paulo, sp, brazil). the manovacuometer was connected to an inextensible rubber hose of 38 cm in length and 0.5 cm in diameter ; the hose was connected to a mouthpiece with a 2-mm hole to avoid any influence of intra - oral pressure on the measurements. a nose clip was used to avoid air leakage through the nostrils. for the pimax measurement, the patients began at the residual volume level and performed a maximal inspiration until total lung capacity was reached. for the pemax measurement, the patients began at total lung capacity and performed a maximal expiration until only residual volume remained. maximal pressures were expected to be sustained for at least one second. for each measurement, data distribution was verified by the shapiro - wilk test. only fev1/fvc (%) and fmi presented non - parametric distributions. the groups were compared by one - way anova followed by the sheff post hoc, or, when necessary, by the kruskal - wallis test followed by multiple comparisons (z values). these f to enter and remove values were chosen in order to detect variables that were significant at p < 0.05. the walking distance, expressed as a percentage value, was considered as the dependent variable, and only the variables that were significantly associated with the walking distance in pearson s correlations were used in the multiple regression analyses. the results are presented as mean standard error of the mean (sem). a p - value lower than 0.05 was considered significant in two - tailed analyses. all analyses were performed using statistica version 7 software (statsoft, tulsa, ok, usa). the spirometry, anthropometry, body composition, exercise capacity and respiratory muscle strength results are presented in table 1, which is organized by the patients nutritional status. there were no statistically significant differences among the groups in age, height, fev1, fev1/fvc or mvv. however, the overweight / obese group had significantly higher body weight, bmi, ffmi, fmi, walking distance (449 36 m vs. 410 32 m vs. 280 37 m, p = 0.02 ; overweight vs. normal weight vs. underweight, respectively) and pimax compared to the normal weight and underweight groups. moreover, the normal weight group had higher weight, bmi and ffmi in comparison to the underweight group. it is worth noting that the power for the anova comparisons presented in table 1 was as follows : age = 0.19, fev1 = 0.42, fev1/fvc = 0.64, mvv = 0.42, weight = 1.00, height = 0.42, bmi = 1.00, ffmi = 1.00, walking distance = 0.87, pimax = 0.91, pemax = 0.26. the correlations between walking distance and age, spirometry, body composition and respiratory muscle strength are presented in table 2. there were significant correlations between walking distance and fev1/fvc, weight, bmi, ffmi and pimax. therefore, these variables were used in a backward stepwise multiple linear regression analysis, which showed that ffmi was the unique significant independent predictor of walking distance (partial r = 0.52, p < 0.001) (table 3). the correlation between walking distance and ffmi the few previous studies that present data on the influence of being overweight or obese on the exercise capacity of patients with severe copd report conflicting results.17,20 therefore, the present study aimed to investigate the impact of nutritional status on the body composition, exercise capacity and respiratory muscle strength of patients with severe copd. we found that the overweight / obese patients had greater ffm, exercise capacity and respiratory muscle strength and that ffm was the main predictor of exercise capacity. the higher exercise capacity found in this study in patients with severe copd who were overweight / obese is supported by the results of ischaki el al.,17 celli.8 and cote.,21 who included overweight and obese patients in their samples but did not analyze overweight and obese subjects separately from normal weight subjects. therefore, it is important to analyze the association between nutritional status and exercise capacity taking into account the contributions of ffm and fm separately. in this context, vanitallie.26 proposed expression of ffm and fm normalized to the square of the height the ffmi has been shown to be more useful than the bmi in identifying subjects with protein - energy malnutrition, and it has been shown to be more accurate for classifying disease severity17 and predicting mortality in patients with copd.6 in this context, the data obtained by our bioimpedance measurements demonstrated that overweight / obese patients presented higher ffmi and fmi in comparison to normal weight and underweight patients. this result is similar to that of poulain.,31 who showed that copd patients who are overweight / obese tend to present with higher ffm. in our study, the ffmi was the main significant predictor of exercise capacity in a backward multiple linear regression analysis. indeed, it has previously been shown that patients with normal weight and depleted ffm have lower exercise capacity in comparison to underweight patients with relative preservation of ffm.32 our results suggest that the overweight / obese patients in our study had higher exercise capacity due to their higher ffm. the role of ffm as a determinant of higher exercise capacity might result from its influence on several important factors. because skeletal muscle mass is the major contributor to ffm and because respiratory muscle strength has been shown to affect the exercise capacities of normal and underweight patients with copd,33 we attempted to determine whether differences in respiratory muscle function could explain the observed differences in exercise capacity between the various groups. while we found that obese / overweight patients had higher respiratory muscle strength than patients who were underweight, no variable representative of respiratory muscle function was a significant predictor of exercise capacity. these results suggest that peripheral skeletal muscles may make a more important contribution to exercise capacity than respiratory muscles. however, because peripheral muscle function was not measured in the present study, we can only speculate about this contribution. it is important to note that, for the general population, mechanistic studies, studies regarding metabolic risk factors and studies regarding cardiovascular disease and premature mortality have shown that a high amount of body fat is associated with an increased risk of cardiovascular disease and early mortality.34 despite this evidence, recent studies indicate that exercise capacity can modulate the harm caused by excessive body fat. for example, mcauley. evaluated 12,417 veterans aged 40 to 70 years and showed that overweight and obese men had increased longevity only if they registered high fitness.2 considering that exercise capacity is strongly related to prognosis for several diseases including copd8,21 and that the present study and others8,17,21 have shown that overweight / obese patients present higher exercise capacity than normal weight or underweight patients, the relationship between overweight / obesity and exercise capacity may represent a plausible mechanism to explain why severe copd patients who are overweight or obese have a better prognosis. we found that the overweight / obese patients had a greater ffm and higher respiratory muscle strength. because it has been shown that ffm is an independent predictor of death6 and that respiratory muscle strength is associated with better functional status, both of these factors could also explain why severe copd patients with overweight / obesity have better prognoses. based on high - resolution computed tomographic scanning, loss of ffm appears to be more frequent in patients with emphysema - type copd than in patients with chronic bronchitis.35 we were unable to differentiate copd subtypes in our sample. in addition, because there were few obese patients in our sample, we analyzed them together with overweight patients. it is worth noting that, for the most important comparisons between groups, the statistical analysis of the study data possessed more than 80% power to detect significant differences at a p - value lower than 0.05. because patients with more severe obesity might present with lower exercise capacity, lower respiratory muscle strength and other dysfunctions associated with metabolic syndrome that could lead to a worse prognosis, other studies should attempt to determine the degree of obesity that reflects a better functional status for copd patients. other cohort studies using mortality as an endpoint are necessary to confirm that ffm, exercise capacity and respiratory muscle strength promote longer life in overweight / obese severe copd patients. severe copd patients who were overweight / obese had greater ffm, exercise capacity and inspiratory muscle strength than patients with the same degree of airflow obstruction who were of normal weight or underweight. higher ffm was independently associated with higher exercise capacity. considering that ffm and exercise capacity are independent predictors of death and that respiratory muscle strength is associated with functional capacity, our results might explain, at least in part, why being overweight or obese is often associated with better survival in patients with severe copd. | introduction : being overweight or obese is associated with a higher rate of survival in patients with advanced chronic obstructive pulmonary disease (copd). this paradoxical relationship indicates that the influence of nutritional status on functional parameters should be further investigated.objective:to investigate the impact of nutritional status on body composition, exercise capacity and respiratory muscle strength in severe chronic obstructive pulmonary disease patients.methods:thirty-two patients (nine women) were divided into three groups according to their body mass indices (bmi) : overweight / obese (25 bmi 34.9 kg / m2, n=8), normal weight (18.5 bmi 24.9 kg / m2, n=17) and underweight (bmi < 18.5 kg / m2, n=7). spirometry, bioelectrical impedance, a six - minute walking distance test and maximal inspiratory and expiratory pressures were assessed.results:airway obstruction was similar among the groups (p=0.30) ; however, overweight / obese patients had a higher fat - free mass (ffm) index [ffmi = ffm / body weight2 (meansem : 170.3 vs. 150.3 vs. 140.5 m / kg2, p<0.01) ], exercise capacity (908 vs. 796 vs. 578 m, p=0.02) and maximal inspiratory pressure (637 vs. 575 vs. 358 % predicted, p=0.03) in comparison to normal weight and underweight patients, respectively. in addition, on backward multiple regression analysis, ffmi was the unique independent predictor of exercise capacity (partial r=0.52, p<0.01).conclusions : severe chronic obstructive pulmonary disease (copd) patients who were overweight or obese had a greater ffm, exercise capacity and inspiratory muscle strength than patients with the same degree of airflow obstruction who were of normal weight or underweight, and higher ffm was independently associated with higher exercise capacity. these characteristics of overweight or obese patients might counteract the drawbacks of excess weight and lead to an improved prognosis in copd. |
schwannoma is a benign nerve sheath tumor originating from schwann cells, and is known as neurilemmoma and neurinoma. although the head and neck region is a rather common site for these benign lesions, intraoral schwannomas, especially the intraosseous types, are very rare, and can account for less than 1% of the benign intraosseous neoplasms. the clinical and radiographic findings of intraosseous schwannoma may be similar to many other lesions such as odontogenic cysts and other benign tumors. therefore, various differential diagnoses have been proposed, which may lead to some sort of confusion in its diagnosis. according to the observation in the literature, these lesions are more common in the mandible and among females, which are mostly observed in the 2 and 3 decades of life. here, along with a review of the related literature, we try to present a case of intraosseous schwannomas that has been recognized in a 9-year - old boy. the patient was a 9-year - old boy who was referred to the department of oral and maxillofacial pathology of the isfahan university of medical science 5 months ago, while complaining about a swelling in the inferior border of the right mandible. deviation of anterior teeth and looseness of teeth (d, e) were seen. in the panoramic radiograph, radiolucency with sclerotic border from mesial of tooth 36 to distal of 3c and transposition of first and second impacted premolar was observable [figure 1 ]. radiolucency with sclerotic border from mesial of tooth 36 to distal of 3c in the panoramic radiogeraph axial computed tomography scan revealed a unilacular lesion in the left mandibular body, 2.5 3 cm in size, with a well - defined border, marked expansion and thinning of the buccal and lingual cortical plates [figure 2 ]. unilacular lesion in the left mandibular body, 2.5 3 cm in size, with well - defined border in axial ct scan first, on the basis of clinical and radiographic features, we can suggest the following lesions : keratocystic odontogenic tumor, central giant cell granuloma, unicystic ameloblastoma and ameloblastic fibroma. excisional biopsy was performed and irregular masses of solid tissues were observable during biopsy. in the histopathology features, ovoid to spindle shape and epithelioid cells were identified, some of which indicated slight pleomorphism and hyperchromatism besides, some regions showed intracellular edema and degenerative changes. in some parts, numerous blood vessels and perivascular hyalinization antoni a : palisading arrangement of spindle cell, verocay body, antoni b : spindle cells haphazardly distributed in a delicate fibrillar matrix. h and e : original maginifiction (40) based on histopathologic features, several differential diagnoses were first taken into account : benign fibroblastic tumor, benign neural tumors and neoplastic vascular lesions. in order to reach final diagnosis, immunohistochemical staining was carried out. the findings revealed that the intensity of s100 staining was 4 + and those of ki67 was weak and 10% at most [figure 4 ]. according to these studies and immunohistochemistry, and regarding clinical and radiographic features and hematoxylin and eosin, in which there were some regions similar to antoni a with palisading cells and similarities of some regions to antoni b, we reached the final and definite diagnosis of the lesion. original maginifiction (400) after 4 months, the patient was evaluated by taking panoramic radiography, and it seems that reparative process is running within the lesion [figure 5 ]. although schwannoma is a benign and slow growing tumor, and it is usually an asymptomatic one, almost 50% of the cases have been reported with pain. the most common site of this tumor in the head and neck region is the tongue. moreover, the intraosseous type is extremely rare, and accounts for less than 1% of all primary bone tumors. average age of the people afflicted is 34 years, and the male to female ratio is 1:1.5. lesions are more common in the mandible than in the maxilla, and the swelling usually involves the body of the mandible. so far, fewer than 50 cases have been recorded in the related literature, of which 44 cases have been reported by chi. in this report, the clinical features of these cases have been described, and only one case was younger than 10 years old. our case is a 9-year - old boy, which appears to be extremely rare. schwannoma can be originating from all cranial nerves, with the exception of the olfactory and optic nerves. the tumor presents an expansive growth pattern, which may cause dislocation of teeth and may also involve the nerves. these above - mentioned features were also observed in the case under investigation in this study. but, the problem was that due to the scarcity of such cases, it was not possible for us to predict the schwannoma in differential diagnosis at first. in microscopic features, schwannoma is an encapsulated tumor that shows two microscopic patterns : antoni a and antoni b. streaming fascicles of spindle - shaped schwann cells characterize antoni a tissue. these cells often form a palisaded arrangement around the central acellular, eosinophilic areas, identified as verocay bodies. antoni b pattern is less cellular and the spindle cells are randomly arranged within a loose, myxomatous stroma. schwannoma indicates the positivity of s100, cd34 and ema only in capsules of the tumor. in our case the lesion showed tumoral appearance, which was completely different from the characteristic structures of odontogenic cysts or tumors. also, specific types of cell, such as giant cells, were not obvious. the lesion showed spindle cell pattern in most of the places, which was similar to fibroblastic or neural cells. immunohistochemistry revealed the tumor to be positive for the s100 protein, confirming nerve origin. on the basis of schwannoma, two characteristic patterns : observing typical limited area similar to antoni a despite verocay body was not clearly seen, and other regions akin to antoni b pattern, the diagnosis of schwannoma was made. the present of these two important histopathologic patterns, lack of mast cells, which is one of the most helpful features in neurofibroma and also diffuse, strongly positively tumoral cells s100 expression in ihc slides, the diagnosis of nurofibroma was rejected. it is probable that some malignant changes in schwannoma may happen, even though some malignant cases have been reported. these changes consist of cyst formation, necrosis, hyalinization, hemorrhage, hemosiderin deposits, inflammation, fibrosis and nuclear atypia. nonetheless, these tumors are benign and atypical mitosis are never seen, and the pathologist must be cautious not to mistake these changes for evidence of a malignancy. also, it is possible to remove the involved nerve in order to decrease the risk of recurrence. because this lesion can be misdiagnosed clinically, radiographically and microscopically as periapical lesion, ameloblastoma, myxoma and central giant cell granuloma, careful attention should be directed toward reaching the diagnosis. only then can the appropriate treatment can be obtained. | intraosseous schwannomas is a very rare neoplasm, and less than 50 cases have been reported in the medical literature. in this article, the clinical, radiographic and histopathologic appearances of a rare case of intraosseous schwannomas are presented. the importance of this case is that other benign central lesions such as odontogenic tumors and cysts might be included in differential diagnosis. this case was recognized in a 9-year - old child, which is a very rare occurrence. the diagnosis was confirmed by immunohistochemical staining with s100 protein. |
a 73-year - old male presented to the medical assessment unit with sudden onset of a 2-day history of severe left - sided headache. he described the headache as dull and throbbing, predominantly localized in the frontal and temporal areas on the left side radiating down to the left side of the neck. on further examination in addition, the patient had mild nausea and slight photophobia since the headache began. on physical examination, he was afebrile and vital signs were stable ; however, he had an exquisitely tender left temporal artery. a complete vascular examination was performed, which did not reveal any upper extremity pulse loss or subclavian, carotid, and axillary bruits. there was no asymmetry of pulses and blood pressure in the extremities. computed tomography of the head ruled out any space - occupying lesions or hemorrhagic lesions. past medical history included well - controlled chronic obstructive airway disease with good exercise tolerance for independent daily activities. a full blood count showed a normal white cell count of 9.210/l with only positive findings of a raised erythrocyte sedimentation rate (esr) of 27 mm / h (normal range : 115 mm / h) and a slightly raised c - reactive protein of 10 mg / l (normal range : 50 years old.2 the disease is unlikely to occur in individuals 50 years3 and incidence of 2.2 per 10,000 patient - years in the uk.4 the mean age for gca occurrence is 72 years,2 as gca is a type of systemic vasculitis whose symptomatology is quite extensive. the symptoms could include constitutional symptoms such as tiredness, fever, and weight loss. fever is usually of low grade ; however, high - grade fever of > 39c has been reported in 15% of patients.5 other clinical features include abrupt - onset headache, which is usually unilateral affecting the temporal region ; however, diffuse and bilateral headaches have been reported;1,4 scalp pain or localized scalp tenderness ; jaw claudication ; nonproductive cough;6 and visual symptoms such as amaurosis fugax, blurring of vision, or even blindness in severe cases. other associated conditions include polymyalgia rheumatica and, in chronic cases, aortic involvement leading to aneurysms and aortic dissection.2 aortic disease features are usually a consequence of long - term, low - grade vasculitis and typically not present at the time of diagnosis. less common complications may include dysarthria,7 throat pain and tongue infarction,8 mononeuritis multiplex,9 sensorineural hearing loss,10 and, even rarely, mesenteric ischemia.11 according to the american college of rheumatology (acr), diagnosis of gca should be considered in all patients aged > 50 years who have at least three of the five criteria findings (table 1). the gold standard test to confirm the diagnosis is tab, which should be performed in all patients suspected of gca ; however, consideration should be given to the urgency of initiation of medical treatment and waiting for biopsy should not delay initiation of high - dose steroid treatment in a suspected case of gca.1 the positivity of the biopsy results can be variable and is dependent on numerous factors. these include sampling error due to skip lesions, attaining too small a sample (< 2 cm), and different phenotypic disease not associated with cranial arteritis in which case the tab will be negative even if repeated.12 such patients may have gca but only involving large vessels, including subclavian artery (arm claudication), carotid artery, and the aorta (aneurysms and dissection),12 but without cranial vessel involvement. these patients will not require tab ; however, they may need systemic imaging to assess isolated large - vessel gca. early tab should be performed preferably within 12 weeks of initiating glucocorticoids ; however, reports suggest that results may remain positive for 26 weeks following initiation of glucocorticosteroids.13,14 duplex ultrasonography secures a promising role in the future for diagnosis of gca, particularly large - vessel disease ; however, it does not currently replace tab as the first - line investigation as recommended by the british society of rheumatology guidelines.1 it is user dependent and requires a high level of expertise, which are yet not currently widespread in uk. furthermore, it does not have the added prognostic value of histology. in case of large - vessel gca, ultrasound is still a sensitive technique, especially for upper limb vasculitides. historically, esr has been considered one of the most useful markers to predict the likelihood of having gca. a normal esr makes gca unlikely ; however, esr does not rule it out.2 a meta - analysis of 114 studies showed that normal esr values indicate much less likelihood of a positive diagnosis of gca (negative likelihood ratio (lr) for abnormal esr, 0.2 ; 95% confidence interval [ci ], 0.080.51), but physical findings such as temporal artery tenderness indicated a higher likelihood of gca (positive lr, 2.6 ; 95% ci, 1.93.7).2 this meta - analysis revealed that a high level of esr was a less important indicator in ruling out gca as the underlying cause for the patient s symptoms, but positive physical findings, characteristic of gca, were more likely to be a strong indicator of a positive diagnosis of gca. our case reported to have positive clinical findings such as temporal artery tenderness with a mildly raised esr ; however, esr was not raised to fulfill the acr criteria for gca. treatment includes immediate initiation of high - dose glucocorticosteroids, recommended upon suspicion of gca.1 visual loss is an early complication of the disease ; however, once established, it rarely improves, and hence, the emphasis is on early treatment. the total duration of high - dose prednisolone is usually governed clinically by the resolution of symptoms and improvement in laboratory test abnormalities.16,17 the dose is then reduced by 10 mg every 2 weeks up to 20 mg, then by 2.5 mg every 24 weeks up to 10 mg, and then by 1 mg every 12 months, provided there are no further relapses.18 these patients should also be coprescribed bone protection (weekly bisphosphonates and calcium / vitamin d supplementation).19 gastrointestinal protection with proton pump inhibitors should also be used. considering that these patients are on a high dose of glucocorticosteroids, they require close monitoring of adverse events, which can be possible through shared care with primary care physicians. at each visit, the following investigations should be performed:1 full blood count, esr, c - reactive protein, urea and electrolytes, and glucose (to look for steroid - induced diabetes);every 2 years, chest radiograph to monitor for aortic aneurysm (more detailed investigations such as mri and echocardiography may also be appropriate);as patients are on long - term steroid therapy, bone mineral density scans may be required. full blood count, esr, c - reactive protein, urea and electrolytes, and glucose (to look for steroid - induced diabetes) ; every 2 years, chest radiograph to monitor for aortic aneurysm (more detailed investigations such as mri and echocardiography may also be appropriate) ; as patients are on long - term steroid therapy, bone mineral density scans may be required. british society of rheumatology recommend close monitoring at weeks 0, 1, 3, and 6 and then at months 3, 6, 9, and 12 in the first year.1 unscheduled visits are advised in the event of relapse. treatment of relapse follows a similar regimen:1 headache : patients should be restarted on the previous higher prednisolone dosage.headache and jaw claudication : treatment should be started with 60 mg prednisolone.visual symptoms : treat with either 60 mg prednisolone or intravenous methylprednisolone.large-vessel gca : further investigation such as positron emission tomography and mri imaging is recommended and consider treatments using systemic vasculitis protocols. headache : patients should be restarted on the previous higher prednisolone dosage. headache and jaw claudication : treatment should be started with 60 mg prednisolone. large - vessel gca : further investigation such as positron emission tomography and mri imaging is recommended and consider treatments using systemic vasculitis protocols. recent study evaluating risks of cranial ischemic event in gca patients revealed that patients with low systemic inflammatory response (odds ratio [or ] = 0.30, 95% ci, 0.081.08), hypertension (or = 7.77, 95% ci, 0.8372.76), and a past history of ischemic heart disease (or = 8.65, 95% ci, 0.9280.95) are associated with a high risk of developing severe cranial ischemic events.20 low - dose aspirin has also been shown to decrease cranial complications.1 in resistant or recurrent gca, immunosuppressive agents such as methotrexate may be used as adjuvant therapy to allow reduction in glucocorticosteroid use. a meta - analysis identified different relationships between clinical features and tab positivity.2 a positive tab has been associated with cranial complications, including cerebrovascular strokes.2 clinical features that increase the likelihood of having a positive tab include jaw claudication : shows a high positive lr 4.2 (2.86.2), diplopia : lr 3.4 (1.38.6), temporal artery beading : lr 4.6 (1.118.4), and temporal artery tenderness : lr 2.6 (1.93.7). features that have been shown to have less likelihood of a positive tab include the absence of temporal artery abnormality (beading, tenderness) : negative lr 0.53 (0.380.75) and a normal value of esr : negative lr 0.2 (0.080.51).2 at each visit, the following investigations should be performed:1 full blood count, esr, c - reactive protein, urea and electrolytes, and glucose (to look for steroid - induced diabetes);every 2 years, chest radiograph to monitor for aortic aneurysm (more detailed investigations such as mri and echocardiography may also be appropriate);as patients are on long - term steroid therapy, bone mineral density scans may be required. full blood count, esr, c - reactive protein, urea and electrolytes, and glucose (to look for steroid - induced diabetes) ; every 2 years, chest radiograph to monitor for aortic aneurysm (more detailed investigations such as mri and echocardiography may also be appropriate) ; as patients are on long - term steroid therapy, bone mineral density scans may be required. british society of rheumatology recommend close monitoring at weeks 0, 1, 3, and 6 and then at months 3, 6, 9, and 12 in the first year.1 unscheduled visits are advised in the event of relapse. treatment of relapse follows a similar regimen:1 headache : patients should be restarted on the previous higher prednisolone dosage.headache and jaw claudication : treatment should be started with 60 mg prednisolone.visual symptoms : treat with either 60 mg prednisolone or intravenous methylprednisolone.large-vessel gca : further investigation such as positron emission tomography and mri imaging is recommended and consider treatments using systemic vasculitis protocols. headache : patients should be restarted on the previous higher prednisolone dosage. headache and jaw claudication : treatment should be started with 60 mg prednisolone. large - vessel gca : further investigation such as positron emission tomography and mri imaging is recommended and consider treatments using systemic vasculitis protocols. recent study evaluating risks of cranial ischemic event in gca patients revealed that patients with low systemic inflammatory response (odds ratio [or ] = 0.30, 95% ci, 0.081.08), hypertension (or = 7.77, 95% ci, 0.8372.76), and a past history of ischemic heart disease (or = 8.65, 95% ci, 0.9280.95) are associated with a high risk of developing severe cranial ischemic events.20 low - dose aspirin has also been shown to decrease cranial complications.1 in resistant or recurrent gca, immunosuppressive agents such as methotrexate may be used as adjuvant therapy to allow reduction in glucocorticosteroid use. a meta - analysis identified different relationships between clinical features and tab positivity.2 a positive tab has been associated with cranial complications, including cerebrovascular strokes.2 clinical features that increase the likelihood of having a positive tab include jaw claudication : shows a high positive lr 4.2 (2.86.2), diplopia : lr 3.4 (1.38.6), temporal artery beading : lr 4.6 (1.118.4), and temporal artery tenderness : lr 2.6 (1.93.7). features that have been shown to have less likelihood of a positive tab include the absence of temporal artery abnormality (beading, tenderness) : negative lr 0.53 (0.380.75) and a normal value of esr : negative lr 0.2 (0.080.51).2 the diagnosis of temporal arteritis requires a high index of suspicion as it may manifest in a variety of clinical features. however, a mildly elevated esr in the presence of positive characteristic clinical features is increasingly suggestive of gca and should still trigger initiation of treatment for gca. acute medical professionals need to be aware of the criteria of gca but, in addition, need to be aware of atypical gca presentations such as low esr, arm claudication, dysarthria, and phenotypic cases not involving cranial arteries. consider diagnosis of temporal arteritis in patients with new onset or new type of localized headache having age 50 years. consider diagnosis of temporal arteritis even in patients with esr < 50 mmhg / h but who meet the criteria of temporal arteritis clinically. prompt diagnosis and urgent treatment should be initiated by the acute medical doctors for temporal arteritis. | temporal arteritis, also known as giant cell arteritis (gca), is a systemic vasculitis that predominantly involves the temporal arteries. it is a medical emergency and should be treated promptly as it can lead to permanent loss of vision. it is very commonly associated with a raised erythrocyte sedimentation rate (esr), usually > 50 mm / h, one of the essential criteria defined by the american college of rheumatology classification of gca. here, we describe the case of a 73-year - old male presenting with a 2-day history of a sudden onset of a severe left - sided headache, which had the signs and symptoms consistent with gca but he had an esr of only 27 mm / h. the patient was urgently treated with prednisolone 60 mg per day, and his symptoms dramatically improved within 24 hours of therapy. temporal artery biopsy results were consistent with an inflammatory response, and withdrawal of treatment led to a relapse of the symptoms. the patient was slowly tapered off the high steroid dose and is now currently managed on a low steroid dose. we should keep a high index of suspicion for gca in patients presenting with clinical symptoms of gca even though the esr is < 50 mm / h as stated in the criteria for gca diagnosis. |
dental caries is a preventable disease of the mineralised tissues of the teeth with a multifactorial etiology related to the interactions over time between tooth substance and certain microorganisms and dietary carbohydrates producing plaque acids. over the last few decades fluorides have been found to be extremely effective in preventing caries on the smooth surfaces of the teeth, but are less effective on the occlusal surfaces. sealants protect the occlusal surfaces inhibiting bacterial growth and providing a smooth surface, increasing the probability that the surface will stay clean. the complex morphology of the occlusal pits and fissures warrants an ideal site for the retention of bacteria and food remnants, rendering proper oral hygiene maintenance difficult. another factor that is responsible for the high incidence of occlusal caries is the lack of salivary access into the fissures due to surface tension, preventing remineralization and thus lessening fluoride effectiveness at this spot as compared with the smooth surfaces. a precise diagnostic method of detection in case of occlusal incipient caries is still not available. the technique of pit and fissure sealants plays, undoubtedly, a fundamental role in preventing occlusal caries, both in primary and in permanent teeth. glass ionomer sealants present a chemical bond to the dental tissue and have an anticariogenic effect by fluoride release. however, the deficiencies of glass ionomer cements are lack of toughness, early water sensitivity, low abrasion resistance and different retention rates. glass ionomer sealants have poorer retention than composite resin materials, and their effect on caries reduction is equivocal. therefore, glass ionomer sealants are mainly used when it is not possible to use a resin material, for example due to poor patient compliance. resin - based sealants are effective in caries control due to a physical barrier formation, which prevents the metabolic exchange between the fissure microorganisms and the oral environment. a fluoride - releasing pit and fissure resin sealant (helioseal - f) is an effort to combine both the caries preventive effect via fluoride release and a good micromechanical bond with the tooth. hence, a study was conducted to evaluate and compare the retention, anatomical form, marginal discoloration and surface texture of two pit and fissure sealants. thirty children between the ages of 6 and 10 years, who were attending the school health program regularly, had participated in the study. approval from the ethical committee and the parents was obtained prior to the onset of the study. a split - mouth design was used in which the two fissure sealants (helioseal - f, ivoclar vivadent somerset, nj and gc fuji vii, gc corporation) were randomly placed in 60 matched contralateral pairs of permanent molar teeth. age of the patient was between 6 and 10 years.presence of all four caries - free permanent first molars.evidence of an acceptable home dental cleaning regimen.patient cooperation and acceptance for the treatment.absence of class i clinical carious lesion.no prior dental therapy.possibility to get proper isolation with cotton rolls.no fluoride mouth rinse program practiced in the school.no central fluoride water supply in the school / community where the students live. no central fluoride water supply in the school / community where the students live. history of any medical disease that might interfere with the study.long-term regimen of medication that could affect the salivary flow and diet modification.current participation in other studies.history of any adverse reaction to any of the restorative materials used.history of abnormal parafunctional activity.heavy occlusal contacts on the teeth to be restored.patients undergoing fluoride application regimen.highly uncooperative child. long - term regimen of medication that could affect the salivary flow and diet modification. patients undergoing fluoride application regimen. highly uncooperative child. a standard fissurotomy bur (ssw fg-330) was used to widen the occlusal pits and fissures of the permanent first molars. the enamel was conditioned by etching with 3537% phosphoric acid and then washed and dried carefully to obtain a chalky - white enamel surface. manufacturer 's instructions were consulted for recommended etch and rinse times. a minimum amount of sealant that was required to adequately cover the pit and fissure network was applied. the restoration was checked for high points using articulating paper. a standard fissurotomy bur (ssw fg-330) fuji vii was mixed according to the manufacturer 's instructions and placed into the prepared tooth and left untouched for 4 min after applying a protective coat of fuji varnish with the help of a microbrush to provide protection against moisture. sealants were rated by a single trained and calibrated examiner using the mouth mirrors and probes following the us public health service criteria. the reason for opting for this criterion was due to its simplicity, easy to record the data in a presentable form and easy communication. scoring was done either by denoting alphabets (like a, b, c etc.) or numerical values (0, 1, 2 etc.). because numerical value was easier for statistical analysis, we have adopted the same in our study. for criteria like anatomical form and marginal discoloration, we have given a score of 0 to indicate acceptability and scores of 1 and 2 to indicate progressively lessening degrees of clinical acceptance. for criteria like surface texture, we have given a score of 0 to indicate acceptability and scores of 1 to indicate progressively lessening degrees of clinical acceptance. the retention was evaluated by visual inspection with the help of a probe and mouth mirror by a single operator as advocated by horowtiz, heifetz and poulsen. a score of 0 was given for complete retention, 1 was given for partial retention and 2 was given for no retention. thirty children between the ages of 6 and 10 years, who were attending the school health program regularly, had participated in the study. approval from the ethical committee and the parents was obtained prior to the onset of the study. a split - mouth design was used in which the two fissure sealants (helioseal - f, ivoclar vivadent somerset, nj and gc fuji vii, gc corporation) were randomly placed in 60 matched contralateral pairs of permanent molar teeth. age of the patient was between 6 and 10 years.presence of all four caries - free permanent first molars.evidence of an acceptable home dental cleaning regimen.patient cooperation and acceptance for the treatment.absence of class i clinical carious lesion.no prior dental therapy.possibility to get proper isolation with cotton rolls.no fluoride mouth rinse program practiced in the school.no central fluoride water supply in the school / community where the students live. history of any medical disease that might interfere with the study.long-term regimen of medication that could affect the salivary flow and diet modification.current participation in other studies.history of any adverse reaction to any of the restorative materials used.history of abnormal parafunctional activity.heavy occlusal contacts on the teeth to be restored.patients undergoing fluoride application regimen.highly uncooperative child. long - term regimen of medication that could affect the salivary flow and diet modification. a standard fissurotomy bur (ssw fg-330) was used to widen the occlusal pits and fissures of the permanent first molars. the enamel was conditioned by etching with 3537% phosphoric acid and then washed and dried carefully to obtain a chalky - white enamel surface. manufacturer 's instructions were consulted for recommended etch and rinse times. a minimum amount of sealant that was required to adequately cover the pit and fissure network was applied. a standard fissurotomy bur (ssw fg-330) was used to widen the occlusal pits and fissures of the permanent first molars. fuji vii was mixed according to the manufacturer 's instructions and placed into the prepared tooth and left untouched for 4 min after applying a protective coat of fuji varnish with the help of a microbrush to provide protection against moisture. sealants were rated by a single trained and calibrated examiner using the mouth mirrors and probes following the us public health service criteria. the reason for opting for this criterion was due to its simplicity, easy to record the data in a presentable form and easy communication. scoring was done either by denoting alphabets (like a, b, c etc.) or numerical values (0, 1, 2 etc.). because numerical value was easier for statistical analysis, we have adopted the same in our study. for criteria like anatomical form and marginal discoloration, we have given a score of 0 to indicate acceptability and scores of 1 and 2 to indicate progressively lessening degrees of clinical acceptance. for criteria like surface texture, we have given a score of 0 to indicate acceptability and scores of 1 to indicate progressively lessening degrees of clinical acceptance. the retention was evaluated by visual inspection with the help of a probe and mouth mirror by a single operator as advocated by horowtiz, heifetz and poulsen. a score of 0 was given for complete retention, 1 was given for partial retention and 2 was given for no retention. the results were tabulated for retention, marginal discoloration, surface texture and anatomical form of pit and fissure sealants and statistically compared using the chi - square test of significance [tables 14 ] [figures 14 ]. comparison of retention between helioseal - f and glass ionomer fuji vii comparison of marginal discoloration between helioseal - f and glass ionomer fuji vii comparison of anatomical form between helioseal - f and glass ionomer fuji vii comparison of surface texture between helioseal - f and glass ionomer fuji vii distribution of cases of helioseal - f and fuji vii with respect to their retention rates distribution of cases of helioseal - f and fuji vii with no marginal discoloration distribution of cases of helioseal - f and fuji vii with good anatomical form distribution of cases of helioseal - f and fuji vii with smooth surface texture taking into consideration a developing country like india, the preventive measures toward oral health are imperative. even if the initial cost of preventive measures like sealants may be higher than the cost of restorative materials, in the long term, sealants or any other preventive measure would be more cost - effective as the tooth would be maintained in a state of health. helioseal - f is shaded white and comprises 40% inorganic filler, including a fluorosilicate glass that slowly releases fluoride ions over time. clinically, helioseal - f forms a smooth surface after polymerization that is easy to clean and does not allow bacteria to settle. other advantages are lack of air bubbles, easy application and simple post - polymerization finishing. fuji vii has a pink shade when set, and this is in contrast from the tooth structure thus enabling inspection for sealant retention. the unique feature of this material is the absence of any resin component in the material to hasten the setting reaction. another major advantage of using glass ionomer vii over glass ionomer materials is the fluoride release by the sealant, which is considered to be the highest among all glass ionomers. in case of partially erupted permanent molars that are prone to caries, their location and gingival covering present difficulties in cleaning and consequently might lead these teeth to become carious before they are fully erupted. effectiveness of sealant may be jeopardized by the difficulty in obtaining ideal isolation and management of tissue during its application. glass ionomer fuji vii has a great advantage that can be applied in areas of minimal isolation, unlike resin - based sealants, where strict isolation and dry field is of utmost importance. several authors showed that the caries increment is low when there is full retention of the sealant.[810 ] in the present study, the 3-month evaluation for the lower arch showed 86.7% of retention for resin sealant and 66.7% retention for glass ionomer sealant. however, the 6-month evaluation showed 83.3% retention for resin sealant and 56.7% retention for glass ionomer sealant, which was in accordance with other studies. the high retention rate reported in this study for resin sealant may be due to the fact that it is easy to apply, good flow, working time is unlimited, chance of air bubble incorporation is less and no mixing is required. they exhibit low technique sensitivity and good adherence in addition to the fluoride releasing property. the glass ionomer acts as a reservoir from which the added fluoride is gradually released into the oral cavity to inhibit enamel demineralization and enhance remineralization. according to wendt and koch, if some part of the sealant is missing in the fissures, there is still enough material in the deeper part to prevent caries. because of the inherent properties of glass ionomer sealants like fluoride release and adherence to dental structures, total or partial loss is not considered to be a problem, as a small amount of material remaining in the fissures results in cariostatic effect.[1517 ] considering that fluoride release by glass ionomer fuji vii is supposed to be six - times the release of fluoride than any other glass ionomer, we can say that glass ionomer fuji vii does show a promising future in the usage as pit and fissure sealant. traditional conventional glass ionomer when used as a pit and fissure sealant shows very poor retention rates when fully retained.[1820 ] a study by booksman., a comparison of 6 months complete retention rates of 92% for concise white light initiated sealant and 2% for the fuji iii glass ionomer sealant, suggested that the routine use of fuji iii glass ionomer as a fissure sealant is unreliable. in the present study, fuji vii had a retention rate of 56.7% and 70% in the lower and upper arch, respectively, at the 1-year evaluation period. the importance of this parameter lies on the fact that an increased surface texture provides a niche for accumulation of plaque and food debris. this can initiate secondary caries at the margins of the sealant. in the present study, this was significant from the 6-month evaluation itself, which can be attributed to the low wear strength of the glass ionomer sealant to occlusal forces. this leads to a faster surface disintegration, thinning of the sealant and, eventually, fracturing it off from the enamel surface. the combination of these two factors seemed to be the main reason for the glass ionomer sealant loss. further improvements on the glass ionomer sealants should be encouraged in order to produce a more wear - resistant product to withstand the occlusal forces. marginal discoloration of a restoration can be considered as an early indicator of its loss of marginal integrity with the adjacent tooth structure. a restoration discolors at its margins when there is marginal breakdown, which creates a rough and irregular surface. this can act as a niche for the accumulation of plaque and food debris and also promote the penetration of oral fluids and cause microleakage, which can lead to secondary caries formation. if there is marginal discoloration that penetrated the sealant margins deep in a pulpal direction, it should be checked thoroughly for any secondary caries, preferably with a radiograph. in the present study, marginal discoloration was checked visually with the help of a mirror. at baseline, all the sealants the cavosurface marginal discoloration was not statistically significant in the 6 months and 1 year evaluation. the marginal discoloration of helioseal - f was higher in the lower arch than in the upper arch. thus, cavosurface margin discoloration is vital for the sealant as this could be the earliest indicator for the initiation of secondary caries. the marginal discoloration of fuji vii sealants was similar in the maxillary and mandibular teeth. when sealants are applied in high caries risk children, review of sealant retention should be part of the recall visit. an earlier guideline in this series has recommended that the recall interval for high caries risk children should not exceed 12 months. if there is particular concern about sealant retention, e.g. if isolation has been difficult to achieve or the sealant has been applied over a suspicious lesion, recall within 6 months is appropriate. a 3-year sealant study involving children aged 514 years with partially or newly erupted first or second permanent molars found that the re - treatment rate was higher at the first 6-month recall than at any other recall during the study, irrespective of the method of tooth isolation used (rubber dam or cotton rolls). the present study suggested that helioseal - f sealant was better than glass ionomer fuji vii sealant with respect to retention, anatomical form and surface texture. the helioseal - f sealant performed better in the upper arch than in the lower arch with respect to all properties (except post - operative sensitivity). the fuji vii sealant performed better in the upper arch than in the lower arch with respect to retention, anatomical form and surface texture. but, with respect to marginal discoloration and post - operative sensitivity, fuji vii showed similar results both in the upper and in the lower arches. more long - term studies are necessary, nevertheless, to determine the potential benefits of both materials. | objective : to evaluate and compare the retention, marginal discoloration, surface texture and anatomical form of pit and fissure sealants.materials and methods : thirty children between the ages of 6 and 10 years, who were attending the school health program regularly, had participated in the study. a split - mouth design was used in which the two fissure sealants (helioseal - f and glass ionomer fuji vii) were randomly placed in 60 matched contralateral pairs of permanent molar teeth. sealants were rated by a single trained and calibrated examiner using mouth mirrors and probes following the us public health service criteria. the sealants were evaluated at 3 months, 6 months and 1 year intervals.results:the data obtained for retention, marginal discoloration, surface texture and anatomical form of pit and fissure sealants were tabulated and compared statistically using the chi - square test of significance.conclusion:the helioseal - f sealant was better than the glass ionomer fuji vii sealant with respect to retention, anatomical form and surface texture. both the materials showed similar results with respect to marginal discoloration. |
the following drosophila stocks were used in the experiments : domeless - gal4 (s.noselli), peroxidasin - gal4 (p.martin), hml - gal4 uas-2xegfp (s.sinenko), dilp2-gal4 (e.rulifson), lsp2-gal4 (c.antoniewski), antp - gal4/tm3,sb (s.cohen) and elav - gal4, mef2-gal4 (bloomington stock center) are gal4 drivers used in this study. we obtained uas - rpr, yw ; p{uas - tor }, w ; p{uas - rheb.pa }, w ; p{uas - wg }, yw ; p{uas - nachbac }, inr / tm2, inr(3r)gc25, inr / tm3,sb, yw;p{uas - inr }, yw ; p{uas - inr }, w p{uas - bsk }, p{tub - gal80},w/fm7c, p{hs - flp }, w ; p{neofrt}82b p{ubi - gfp}83, p{neofrt}82b from bloomington stock center. w ; ilp2, w ; ilp3 and w ; ilp5 were isolated by l. partridge s lab and obtained from bloomington stock center. y, w ; p{uas - dilp2 } was a kind gift from e.hafen (ets - imsb, switzerland), y, w;uas - slif, from p.leopold (universite de nice, france), df[dilp1 - 5]/tm6b, tb and df[dilp1 - 3]/tm6b, tb from l.pick (university of maryland, usa), y, w;tor / sm6b - tm6b and y, w;tor / sm6b - tm6b from t.p.neufeld (university of minnesota, usa), p{msn - gfp.f9 } and eater - gfp from r.a.schulz (university of notre dame, usa), and d4-lacz from a.courey (ucla), drs - gfp from j - m. crosses were maintained at 25c, except for rnai and gal4/uas expression used experiments, for which crosses were maintained at 29c. for gal80 experiments, for synchronization of larvae, eggs were collected for an hour and newly hatched larvae were transferred onto fresh food plates after 24h and aged for specified time periods at 25c. for inr clones in the lymph gland, we generated frt82b inr / tm6b tb, and confirmed the recombinant with ey - flp. larvae containing hs - flp;frt82b inr / frt82b ubi - gfp were heat - shocked at 38c for 40 min in each instar. anterior fat bodies were gently dissected, and fixed with 3.7% formaldehyde for 20 minutes. after visualizing dna with topro3, samples were mounted in vectashield (vector labs). for anti - wg antibody, the following antibodies were used in this study : anti - pxn (1:1500, j.fessler and l.fessler), anti - phosphoakt (1:200, cell signaling # 4054), anti - wg (1:10, dshb), anti - l1 (1:100, i.ando), mouse anti-gal (1:200, promega), anti - dilp2 (1:1000, p. leopold) and anti - ppo (1:250, h.muller). images were obtained using a zeiss lsm700 confocal system using either 20 or 40 immersion oil objective. images were processed using image j (nih). to estimate the expression of differentiation markers within the mz, the middle 3 optical sections from the z stack were merged into a single section. for wild type, this enables the clearest view of the medullary zone surrounded by the differentiating cells of the cortical zone. depending on the genotypes studied, the merged image may contain images at two or three different wavelength channels indicating different markers used for the lymph gland analysis. for the zone specific markers (pxn, hml and dome), the colored areas were recalibrated into an identical threshold by using the binary tool (process - binary - make binary) as an automatic routine. the area with identical threshold was automatically captured with the wand tool, and the size was measured using the measure tool (analyze - measure). to measure total size of the lobe, topro3 expressing area percentage of differentiation was calculated by dividing size of the marker - expressing area by the total size of the lobe (topro3 area). ten randomly selected lymph glands were analyzed per genotype, and statistical significance was calculated with standard t test. to quantify the expression of wingless in different genotypes, we followed the protocol from (http://sciencetechblog.files.wordpress.com/2011/05/measuring-cell-fluorescence-using-imagej.pdf). for hemocyte collection, staged larvae were washed with water and bled in 20l of schneiders insect media. the cells were gently redistributed onto 5 mm 14-well slides (fisher scientific), allowed to settle down for 15min at room temperature, and fixed with 3.7% formaldehyde for 20min. topro3 was used for visualizing dna. for hemocyte counting, cells prepared in 20l schneiders medium were transferred to a neubauer improved hemocytometer, and counted and analyzed as previously described. larvae were synchronized and allowed to grow at 25c until early third instars (9096h after egg deposition (aed)). staged larvae were collected and washed with water, and finally rinsed with ultrapure distilled water (gibco). these larvae were then divided into two groups ; one group of larvae was placed on regular food media (control) and the second was placed on 1% soft agar media (starvation). those raised on agar plates were also maintained with or without yeast to control for any possible effects due to handling and growth on agar plates. per 60 mm dish, 1520 larvae were used. both the standard food media and 1% agar media the larvae were reared for 24 hours at 25c after which they were collected, dissected and processed for antibody staining. for aseptic starvation, collections of embryos were sterilized by rinsing in 0.25% clorox, 0.04% n - alkyl dimethyl benzyl ammonium chloride, washed twice in 70% ethanol following two brief washes in ultra pure distilled water (gibco) and autoclaved distilled water, and then transferred onto axenic food plates. the animals were staged to 9096h aed, and larvae were selected with sterilized forceps and washed with ultrapure distilled water (gibco). normally fed larvae were reared on axenic food media with antibiotic - antimycotic solution, and starved larvae, on sterilized 1% agar plate with antibiotic - antimycotic solution as the standard starvation protocol. both food / agar and homogenized larvae from each plate were plated onto antibiotic - free bacterial lb agar plate, and assessed for lack of growth to verify axenic conditions. no bacterial colony grew with food / agar or homogenized larvae grown in axenic culture conditions. for nutrient supplementation assays, carbohydrate complexes, lipid complexes, and amino acids were added to sterilized 1% agar solution and processed for standard starvation method as described. for yeast supplementation, 34g / l of inactivated yeast powder was added to 1% agar solution, and sterilized, processed for standard starvation method as described above. for rapamycin treatment, 9096h aed larvae were transferred to standard fly medium containing 2 m rapamycin and kept for 24 hours. for anti - inr probe, a 205 bp cdna fragment from ld06045 (gdrc, cdna library) was amplified with inr - for (cga tgg cgg tgt tat gga gag) and inr - rev primers (cgc tcc ttt tcc cga tgc tgc aga). the following drosophila stocks were used in the experiments : domeless - gal4 (s.noselli), peroxidasin - gal4 (p.martin), hml - gal4 uas-2xegfp (s.sinenko), dilp2-gal4 (e.rulifson), lsp2-gal4 (c.antoniewski), antp - gal4/tm3,sb (s.cohen) and elav - gal4, mef2-gal4 (bloomington stock center) are gal4 drivers used in this study. we obtained uas - rpr, yw ; p{uas - tor }, w ; p{uas - rheb.pa }, w ; p{uas - wg }, yw ; p{uas - nachbac }, inr / tm2, inr(3r)gc25, inr / tm3,sb, yw;p{uas - inr }, yw ; p{uas - inr }, w p{uas - bsk }, p{tub - gal80},w/fm7c, p{hs - flp }, w ; p{neofrt}82b p{ubi - gfp}83, p{neofrt}82b from bloomington stock center. w ; ilp2, w ; ilp3 and w ; ilp5 were isolated by l. partridge s lab and obtained from bloomington stock center. y, w ; p{uas - dilp2 } was a kind gift from e.hafen (ets - imsb, switzerland), y, w;uas - slif, from p.leopold (universite de nice, france), df[dilp1 - 5]/tm6b, tb and df[dilp1 - 3]/tm6b, tb from l.pick (university of maryland, usa), y, w;tor / sm6b - tm6b and y, w;tor / sm6b - tm6b from t.p.neufeld (university of minnesota, usa), p{msn - gfp.f9 } and eater - gfp from r.a.schulz (university of notre dame, usa), and d4-lacz from a.courey (ucla), drs - gfp from j - m. crosses were maintained at 25c, except for rnai and gal4/uas expression used experiments, for which crosses were maintained at 29c. for gal80 experiments, for synchronization of larvae, eggs were collected for an hour and newly hatched larvae were transferred onto fresh food plates after 24h and aged for specified time periods at 25c. for inr clones in the lymph gland, we generated frt82b inr / tm6b tb, and confirmed the recombinant with ey - flp. larvae containing hs - flp;frt82b inr / frt82b ubi - gfp were heat - shocked at 38c for 40 min in each instar. lymph glands were dissected and stained as previously described. for fat body samples, anterior fat bodies were gently dissected, and fixed with 3.7% formaldehyde for 20 minutes. after visualizing dna with topro3, samples were mounted in vectashield (vector labs). for anti - wg antibody, the following antibodies were used in this study : anti - pxn (1:1500, j.fessler and l.fessler), anti - phosphoakt (1:200, cell signaling # 4054), anti - wg (1:10, dshb), anti - l1 (1:100, i.ando), mouse anti-gal (1:200, promega), anti - dilp2 (1:1000, p. leopold) and anti - ppo (1:250, h.muller). images were obtained using a zeiss lsm700 confocal system using either 20 or 40 immersion oil objective. images were processed using image j (nih). to estimate the expression of differentiation markers within the mz, the middle 3 optical sections from the z stack were merged into a single section. for wild type, this enables the clearest view of the medullary zone surrounded by the differentiating cells of the cortical zone. depending on the genotypes studied, the merged image may contain images at two or three different wavelength channels indicating different markers used for the lymph gland analysis. for the zone specific markers (pxn, hml and dome), the colored areas were recalibrated into an identical threshold by using the binary tool (process - binary - make binary) as an automatic routine. the area with identical threshold was automatically captured with the wand tool, and the size was measured using the measure tool (analyze - measure). to measure total size of the lobe, percentage of differentiation was calculated by dividing size of the marker - expressing area by the total size of the lobe (topro3 area). ten randomly selected lymph glands were analyzed per genotype, and statistical significance was calculated with standard t test. to quantify the expression of wingless in different genotypes, we followed the protocol from (http://sciencetechblog.files.wordpress.com/2011/05/measuring-cell-fluorescence-using-imagej.pdf). for hemocyte collection, staged larvae were washed with water and bled in 20l of schneiders insect media. the cells were gently redistributed onto 5 mm 14-well slides (fisher scientific), allowed to settle down for 15min at room temperature, and fixed with 3.7% formaldehyde for 20min. cells prepared in 20l schneiders medium were transferred to a neubauer improved hemocytometer, and counted and analyzed as previously described. larvae were synchronized and allowed to grow at 25c until early third instars (9096h after egg deposition (aed)). staged larvae were collected and washed with water, and finally rinsed with ultrapure distilled water (gibco). these larvae were then divided into two groups ; one group of larvae was placed on regular food media (control) and the second was placed on 1% soft agar media (starvation). those raised on agar plates were also maintained with or without yeast to control for any possible effects due to handling and growth on agar plates. per 60 mm dish, 1520 larvae were used. both the standard food media and 1% agar media the larvae were reared for 24 hours at 25c after which they were collected, dissected and processed for antibody staining. for aseptic starvation, collections of embryos were sterilized by rinsing in 0.25% clorox, 0.04% n - alkyl dimethyl benzyl ammonium chloride, washed twice in 70% ethanol following two brief washes in ultra pure distilled water (gibco) and autoclaved distilled water, and then transferred onto axenic food plates. the animals were staged to 9096h aed, and larvae were selected with sterilized forceps and washed with ultrapure distilled water (gibco). normally fed larvae were reared on axenic food media with antibiotic - antimycotic solution, and starved larvae, on sterilized 1% agar plate with antibiotic - antimycotic solution as the standard starvation protocol. both food / agar and homogenized larvae from each plate were plated onto antibiotic - free bacterial lb agar plate, and assessed for lack of growth to verify axenic conditions. no bacterial colony grew with food / agar or homogenized larvae grown in axenic culture conditions. for nutrient supplementation assays, carbohydrate complexes, lipid complexes, and amino acids were added to sterilized 1% agar solution and processed for standard starvation method as described. for yeast supplementation, 34g / l of inactivated yeast powder was added to 1% agar solution, and sterilized, processed for standard starvation method as described above. for rapamycin treatment, 9096h aed larvae were transferred to standard fly medium containing 2 m rapamycin and kept for 24 hours. for anti - inr probe, a 205 bp cdna fragment from ld06045 (gdrc, cdna library) was amplified with inr - for (cga tgg cgg tgt tat gga gag) and inr - rev primers (cgc tcc ttt tcc cga tgc tgc aga). | the drosophila lymph gland is a hematopoietic organ in which progenitor cells, which are most akin to the common myeloid progenitor or cmp in mammals, proliferate and differentiate into three types of mature cells plasmatocytes, crystal cells and lamellocytes the functions of which are reminiscent of mammalian myeloid cells1. during the first and early second instars of larval development, the lymph gland contains only progenitors, whereas in the third instar, a medial region of the primary lobe of the lymph gland called the medullary zone (mz) contains these progenitors2, while maturing blood cells are found juxtaposed in a peripheral region designated the cortical zone (cz)2. a third group of cells referred to as the posterior signaling center (psc) functions as a hematopoietic niche3,4. similar to mammalian myeloid cells, drosophila blood cells respond to multiple stresses including hypoxia, infection, and oxidative stress57. however, how systemic signals are sensed by myeloid progenitors to regulate cell fate determination has not been well described. here, we show that the hematopoietic progenitors of drosophila are direct targets of systemic (insulin) and nutritional (essential amino acid) signals, and that these systemic signals maintain the progenitors by promoting wingless (wnt in mammals) signaling. we expect that this study will promote investigation of such possible direct signal sensing mechanisms by mammalian myeloid progenitors. |
antibiotic resistance is one of the most important global health problems, which leads to increased morbidity, mortality, and health care costs. distribution of resistance pathogens to antibiotics differs and depends on time, hospital, and different hospital wards (1). overall, the excessive use of antibiotics leads to the emergence of antimicrobial resistance ; therefore, the rational use of antibiotics leads to control the spread of this resistance. to reduce the emergence of the antibiotic resistance microbes, use of antimicrobials should be monitored by infection control unit in hospitals (2). the rapid increase in multidrug resistant mdr bacterial strains was observed, which may be due to the poor applied programs (3). patients excessive use of antibiotics, whether on their own or within the hospital is a major factor for the emergence of resistant strains (4). the increasing use of antimicrobials in humans, animals, and agriculture has resulted in developing many pathogens resistant to these powerful drugs (5). resistant bacteria are divided according to the group of antibiotics, they are resistant to. conjugation of a plasmid is the most prevalent type for the acquisition of resistance among bacteria (6). recently, the latest resistance mechanisms have resulted in the emergence of mdr bacterial strains (7). from now on, new antimicrobial agents are needed to eliminate the infection resulted from multidrug resistant microbes and potential use against humans during wars (8). controlling the spread of resistance requires the collaboration of several organizations such as veterinary, medical, and public health communities (9). if the microbe is resistant to more than a class of antibiotics, it is considered a multidrug resistance, and the choice of drug against microbe is mainly associated with the knowledge of its sensitivity, to gain better results in the treatment of patients (10). the aim of this study was to isolate, identify, and characterize the prevalence of clinical isolates along with their antimicrobial sensitivity pattern among the patients referring to king khalid hospital, saudi arabia. a total of 428 clinical samples such as pus - swap, urine, blood, suction tip, catheter tip, pleural fluid, bronchial wash, peritoneal fluid, ear swap, and sputum were collected from the patients of king khalid hospital as a part of routine patient care. study (11). in brief, the sample collection started in june 2013 and continued for 8 months. they were collected from 8 medical and surgical wards, including intensive care unit, female medical ward, male medical ward, male surgical ward, maternity and child care, obstetrics ward, pediatric ward, artificial kidney unit, and nursery. samples were plated on blood agar (ba), macconkey agar (mac), cetrimide agar (ca), and nutrient agar (na) (bought from aldrich chemical, milwaukee, wi, usa). we used microscan instrumentation (auto scan-4 and walkaway system) (siemens healthcare diagnostics inc, usa). panels used in the study were microscan dried gram - positive mic / combo, dried gram - positive breakpoint combo, and dried gram - positive i d type 2 or 3. also, microscan dried gram - negative mic / combo panels and dried gram - negative breakpoint combo panels were used. these panels were designed for use in determining agent susceptibility and or identification of the species, level of rapidly growing aerobic and facultative gram - positive cocci, or aerobic and facultative anaerobic gram - negative bacilli. susceptible, intermediate, and resistant isolates were arranged by location in the hospital according to antibiogram results. a total of 300 non - duplicate clinical isolates of selected organisms were collected and each one was accompanied by antibiogram indicating susceptibility and resistance pattern. also, validation test was conducted on 283 isolates, whereas 17 isolates were lost or not purified. the results of this validation test provided a sense of the accuracy of the laboratory method of the susceptibility test in identifying organisms as resistant. in validation test, mics were employed by the standards of antimicrobial susceptibility testing according to clsi guidelines (13). organisms defined as causing major errors were those categorized by microscan as resistant and found to be susceptible by validity testing. organisms defined as causing minor errors were classified as intermediate by validity test and as susceptible or resistant by microscan or classified as intermediate by microscan and as susceptible or resistant by validity testing. antibiotic susceptibility test was done on these isolates to determine the susceptibility of the isolate to an array of antibiotics, which would determine the extent of resistance or sensitivity of the organism to each antibiotic. a total of 428 clinical samples such as pus - swap, urine, blood, suction tip, catheter tip, pleural fluid, bronchial wash, peritoneal fluid, ear swap, and sputum were collected from the patients of king khalid hospital as a part of routine patient care. study (11). in brief, the sample collection started in june 2013 and continued for 8 months. they were collected from 8 medical and surgical wards, including intensive care unit, female medical ward, male medical ward, male surgical ward, maternity and child care, obstetrics ward, pediatric ward, artificial kidney unit, and nursery. samples were plated on blood agar (ba), macconkey agar (mac), cetrimide agar (ca), and nutrient agar (na) (bought from aldrich chemical, milwaukee, wi, usa). we used microscan instrumentation (auto scan-4 and walkaway system) (siemens healthcare diagnostics inc, usa). panels used in the study were microscan dried gram - positive mic / combo, dried gram - positive breakpoint combo, and dried gram - positive i d type 2 or 3. also, microscan dried gram - negative mic / combo panels and dried gram - negative breakpoint combo panels were used. these panels were designed for use in determining agent susceptibility and or identification of the species, level of rapidly growing aerobic and facultative gram - positive cocci, or aerobic and facultative anaerobic gram - negative bacilli. susceptible, intermediate, and resistant isolates were arranged by location in the hospital according to antibiogram results. a total of 300 non - duplicate clinical isolates of selected organisms were collected and each one was accompanied by antibiogram indicating susceptibility and resistance pattern. also, validation test was conducted on 283 isolates, whereas 17 isolates were lost or not purified. the results of this validation test provided a sense of the accuracy of the laboratory method of the susceptibility test in identifying organisms as resistant. in validation test, mics were employed by the standards of antimicrobial susceptibility testing according to clsi guidelines (13). organisms defined as causing major errors were those categorized by microscan as resistant and found to be susceptible by validity testing. organisms defined as causing minor errors were classified as intermediate by validity test and as susceptible or resistant by microscan or classified as intermediate by microscan and as susceptible or resistant by validity testing. antibiotic susceptibility test was done on these isolates to determine the susceptibility of the isolate to an array of antibiotics, which would determine the extent of resistance or sensitivity of the organism to each antibiotic. the study was carried out at the laboratory in the college of applied medical sciences and king khalid hospital over a period of 8 months from june 2013 to january 2014. during the entire study period, of 428 clinical samples, 300 bacterial isolates were obtained, which accounting for an isolation rate of 70.1%. 116 (39.0%) isolates were isolated and identified from wounds, 97 (32.3%) from urine, 54 (17.8%) from blood, 20 (6.7%) from suction tip, 6 (2.0 %) from catheter tip, 2 (0.8%) from purulent discharge, and 1 (0.3%) from each bronchial wash, peritoneal fluid, ear swab, pleural fluid, and sputum (table 1). the culture reports revealed that gram - negative organisms like k. pneumonia, e. coli, and p. aeruginosa were the predominant organisms, (77.3%, n = 232), followed by gram - positive organisms like staphylococcus aureus (22.7%, n = 68) (table 1). data indicated the highest prevalence of e. coli and s. aureus over all other organisms during the study period. isolates of e. coli were found to be 38.3% (n = 115) of all obtained isolates while s. aureus was found to comprise 22.7% (n = 68) isolates. on the other hand, k. pneumonia and p. aeruginosa rates were 21.7% (n = 65) and 17.3% (n = 52), respectively (table 1). also, majority were isolated from pus / swap, urine, and blood samples. a total of 283 non - duplicate clinical isolates containing 4 various microorganisms were underwent susceptibility testing against various antibiotics. resistant and susceptibility profile of s. aureus showed its high resistance to both ampicillin and linezolid (94.1%) and high sensitivity to more than one antibiotic such as daptomycin, penicillin, synercid, teicoplanin, vancomycin, and trimethoprim - sulfamethoxazole, which have sensitivity rate more than 88% (table 2). klebsiella pneumonia was fully resistant to ampicillin (100%) followed by mezlocillin and piperacillin (92.3%) and showed highest sensitivity to imipenem (84.61%) followed by amikacin and piperacillin - tazobactam (76.92%). pseudomonas aeruginosa was fully resistant to 4 antibiotics of cefazoline, cefoxitin, tetracycline and trimethoprim / sulfamethoxazole (100%) (table 3)., e. coli isolates were highly resistant (more than 78%) to several antibiotics such as amikacin, ampicillin, mezlocillin, and piperacillin. also, e. coli isolates had the highest sensitivity (more than 86%) to fosfomycin, imipenem, piperacillin / tazobactam, and tigecycline (table 3). abbreviations : amox - clav, amoxicillin - clavulanic ; pip - tazo, piperacillin - tazobactam ; trimeth - sulfa, trimethoprim - sulfamethoxazole ; nt, not tested. validity test by broth microdilution of 107 amikacin-, ampicillin-, mezlocillin-, or piperacillin - resistant e.coli sent by the hospital revealed 2 minor errors (1.8%) and 2 major errors (1.8%). also, testing 60 ampicillin-, mezlocillin-, or piperacillin - resistant k. pneumonia isolates showed 4 minor errors (6.7%). on the other hand, for validity testing of 64 methicillin - resistant s. aureus (mrsa) or 52 cefazoline-, cefoxitin-, tetracycline-, or trimethoprim - sulfamethoxazole - resistant p. the rate of errors varied slightly between the two test methods ; microscan and broth microdilution susceptibility testing. results also showed increasing number of multiple - drug resistant (mdr) isolates in female surgical, 100% ; icu 90.2% ; male surgical, 88.2% ; obstetrics and nursery, 75.0% ; pediatrics 69.2% ; and artificial kidney wards, 54.2% (table 4). abbreviations : icu, intensive care unit ; nd, the microbe not detected in that area ; trimeth - sulfa, trimethoprim - sulfamethoxazole. of 428 clinical samples, 300 bacterial isolates were obtained, which accounting for an isolation rate of 70.1%. 116 (39.0%) isolates were isolated and identified from wounds, 97 (32.3%) from urine, 54 (17.8%) from blood, 20 (6.7%) from suction tip, 6 (2.0 %) from catheter tip, 2 (0.8%) from purulent discharge, and 1 (0.3%) from each bronchial wash, peritoneal fluid, ear swab, pleural fluid, and sputum (table 1). the culture reports revealed that gram - negative organisms like k. pneumonia, e. coli, and p. aeruginosa were the predominant organisms, (77.3%, n = 232), followed by gram - positive organisms like staphylococcus aureus (22.7%, n = 68) (table 1). data indicated the highest prevalence of e. coli and s. aureus over all other organisms during the study period. isolates of e. coli were found to be 38.3% (n = 115) of all obtained isolates while s. aureus was found to comprise 22.7% (n = 68) isolates. on the other hand, k. pneumonia and p. aeruginosa rates were 21.7% (n = 65) and 17.3% (n = 52), respectively (table 1). also, majority were isolated from pus / swap, urine, and blood samples. a total of 283 non - duplicate clinical isolates containing 4 various microorganisms were underwent susceptibility testing against various antibiotics. resistant and susceptibility profile of s. aureus showed its high resistance to both ampicillin and linezolid (94.1%) and high sensitivity to more than one antibiotic such as daptomycin, penicillin, synercid, teicoplanin, vancomycin, and trimethoprim - sulfamethoxazole, which have sensitivity rate more than 88% (table 2). klebsiella pneumonia was fully resistant to ampicillin (100%) followed by mezlocillin and piperacillin (92.3%) and showed highest sensitivity to imipenem (84.61%) followed by amikacin and piperacillin - tazobactam (76.92%). pseudomonas aeruginosa was fully resistant to 4 antibiotics of cefazoline, cefoxitin, tetracycline and trimethoprim / sulfamethoxazole (100%) (table 3)., e. coli isolates were highly resistant (more than 78%) to several antibiotics such as amikacin, ampicillin, mezlocillin, and piperacillin. also, e. coli isolates had the highest sensitivity (more than 86%) to fosfomycin, imipenem, piperacillin / tazobactam, and tigecycline (table 3). abbreviations : amox - clav, amoxicillin - clavulanic ; pip - tazo, piperacillin - tazobactam ; trimeth - sulfa, trimethoprim - sulfamethoxazole ; nt, not tested. validity test by broth microdilution of 107 amikacin-, ampicillin-, mezlocillin-, or piperacillin - resistant e.coli sent by the hospital revealed 2 minor errors (1.8%) and 2 major errors (1.8%). also, testing 60 ampicillin-, mezlocillin-, or piperacillin - resistant k. pneumonia isolates showed 4 minor errors (6.7%). on the other hand, for validity testing of 64 methicillin - resistant s. aureus (mrsa) or 52 cefazoline-, cefoxitin-, tetracycline-, or trimethoprim - sulfamethoxazole the rate of errors varied slightly between the two test methods ; microscan and broth microdilution susceptibility testing. results also showed increasing number of multiple - drug resistant (mdr) isolates in female surgical, 100% ; icu 90.2% ; male surgical, 88.2% ; obstetrics and nursery, 75.0% ; pediatrics 69.2% ; and artificial kidney wards, 54.2% (table 4). abbreviations : icu, intensive care unit ; nd, the microbe not detected in that area ; trimeth - sulfa, trimethoprim - sulfamethoxazole. contraction of the infection during the health care, often leads to poor prognosis, increase mortality, and health care costs. therefore, execution of an integrated program to reduce the infection (30%) will decline the health care costs (14). studies carried out by different researchers have reported varied isolation rates. in the present study, high isolation rate (70.1%) (16) reported it to be 21.8%. in a study from saudi arabia, eltahawy and khalaf (17) reported 16% of all the gram - negative bacilli isolated. the present study included the types and antibiotic susceptibility pattern of bacterial organisms isolated from different samples of patients in king khalid hospital. the bacteriological methods for isolation and identification resulted in 300 isolates, 116 from wounds, 97 from urine, 54 from blood, 20 from suction tip, 6 from catheter tip, 2 from purulent discharge, and 1 from each of bronchial wash, peritoneal fluid, ear swab, pleural fluid, and sputum. of these isolates, e. coli was the most common with 115 (38.3%) isolates (61 from urine, 39 from wound, 7 from blood, 4 from suction tip, and 1 from each catheter, purulent discharge, peritonea fluid and pleural fluid) followed by s. aureus with 68 (22.7%) isolates (36 isolated from wound, 20 from blood and 12 from suction tip). on the other hand, isolates of k. pneumonia and p. aeruginosa were 65 (21.7%) and 52 (17.3%), respectively. they found that e. coli was the most common organism isolated from pus (47.05%) and its resistant rate was 50.0% followed by s. aureus (29.41%) with the resistant rate of 60.0%. antibiotic susceptibility profile of s. aureus showed its high resistant to ampicillin and linezolid. also, it has high sensitivity to more than one antibiotic, including penicillin, synercid, tetracycline, trimethoprim / sulfamethoxazole, and vancomycin. k. pneumonia isolates were fully resistant to ampicillin (100%) and very sensitive to imipenem (84.61%). pseudomonas aeruginosa was fully resistant to 4 antibiotics : cefazoline, cefoxitin, tetracycline, and trimethoprim / sulfamethoxazole. (22) who reported mdr to 5 antibiotics (ampicillin, chloramphenicol, co - trimoxazole, nitrofurantoin, and tetracycline). multi - resistance p. aeruginosa was also isolated by olowu and oyetunji (24) and fagade. also, aiyegoro. (26), isolated multi - resistance p. aeruginosa in their study to determine the incidence of urinary tract infection in children and adolescents. the higher percentage of mdr isolates from different clinical specimens will become problematic in the future. on the other hand, synercid, teicoplanin, and it showed resistance rate of 5.9% ; low resistance rate of s. aureus may be due to the recent introduction of this antibiotic. our study showed no effective drugs against k. pneumonia and p. aeruginosa, whereas the lowest resistance rates were 15.38% and 36.5% for imipenem. accurate laboratory detection and control of patient to patient transmission are cornerstones in containment of drug resistant. the higher rates of resistance in icu and surgical wards may be parallel with higher usage of antimicrobial drugs. other factors such as use of other drugs or cross - transmission may play an important role in propagation of these organisms. drug resistant may be due to infection control practices, inadequate antibiotic treatment, or noncompletion of treatment course that may lead to infection recurrent and drug resistance. we believe that reporting antimicrobial use must be stratified by hospital wards to make valid comparisons between areas. further studies are required to determine the importance of specific icu type as well as regional variations in the patterns of antimicrobial use. although this surveillance assessed antimicrobial agents in a number of specimens, more research is needed to clarify the reasons of drug resistance and its prevalence in saudi arabia hospitals. | backgroundresearch to understand and control the emergence and spread of antimicrobial resistance has become a public health priority.objectivesthis study was conducted to study epidemiology and resistant pattern of bacteria causing infection in different king khalid hospital units.patients and methodsall samples were sent to the lab and routinely processed according to the standard microbiological procedures. then, the cultures yielding growth were selected for the study. identification and antibiotic susceptibility test for all clinical isolates were processed by using microscan instrumentation. a total of 428 clinical samples were collected within 8 months ; out of them, 300 clinical isolates were subjected to validation test.resultsescherichia coli, klebsiella pneumonia, and pseudomonas aeruginosa were the commonly identified gram - negative bacteria. staphylococcus aureus was the only identified gram - positive bacterium. the most common infections were taken from the wounds (39.0%), urinary tract (32.3%), and bloodstream (17.8%). the most common antibiotic - resistant bacteria were found on female surgical ward (100%) followed by icu (90.2%), and male surgical ward (88.2%). the overall results of antibiotic resistance were 100% for s. aureus, 93.3% k. pneumonia, 75.7 % e. coli, and 100% for p. aeruginosa. staphylococcus aureus showed high resistance to ampicillin and linezolid (94.1%). high (86.95%) and full resistance (100%) against ampicillin were observed from e. coli and k. pneumonia, respectively. p. aeruginosa was fully resistant to 4 antibiotics of cefazoline, cefoxitin, tetracycline, and trimethoprim-sulfamethoxazole.conclusionsthe study was useful in determining the risk factors and defining different hospital units which should be targeted for measures to prevent infection. |
wound - healing in skin and other mucosal tissues involves a complex sequence of events including the clotting cascade, acute and chronic inflammation, reepithelialization, granulation tissue formation, wound contraction, and connective - tissue remodeling. however, a number of genetic and acquired conditions, such as aging, malnutrition (e.g., vitamin c and protein deficiency), infection, hypoxia, and genetic diseases such as ehlers - danlos syndrome, can impair this reparative process. among these conditions, diabetes mellitus is a most common cause of impaired or nonhealing wounds. as an example, the clinical significance of long - term hyperglycemia is highlighted by alarming data showing that 85% of nonhealing the pathway to a chronic wound, as outlined by authors of a recent study, focused on prolonged or chronic inflammation characterized by activation of macrophages (as well as accumulation of neutrophils) that resulted in elevated levels of proinflammatory cytokines, reactive oxygen species, matrix metalloproteinases (mmps), and other neutral proteinases (e.g., elastase). this, coupled with a deficiency of endogenous proteinase inhibitors, all leads to excessive matrix degradation, degradation of growth factors, and impaired epithelialization. in this regard, our discovery three decades ago that the tetracycline antibiotics (including doxycycline), unexpectedly, can inhibit host - derived mmps such as the collagenases (mmp-1, mmp-8, and mmp-13) and gelatinases / type iv collagenases (mmp-2 and mmp-9) and by mechanisms independent of their antimicrobial activity [6, 7 ] resulted in a series of experiments on the effect of these mmp - inhibitors on wound - healing in several different animal models. these studies included the streptozotocin - induced type i diabetic rat and the ovariectomized rat model of postmenopausal osteoporosis. both of these models produce aging - like changes in the collagen of the dermis, including skin atrophy associated with impaired collagen synthesis by fibroblasts, dramatic increases in collagenase and gelatinase activities, and excessive accumulation of older highly cross - linked collagen relative to newer more extractable collagen. all of these contribute, at least in part, to impaired wound - healing in skin [8, 9 ]. using these and other models, we demonstrated that topical or systemic administration of tetracyclines, such as doxycycline, and the chemically modified nonantimicrobial tetracyclines or cmts (aka cols) can normalize wound - healing : (i) while having no effect on the severity of hyperglycemia in the diabetic rats ; (ii) in the presence of prolonged estrogen deficiency in ovariectomized aged rats ; and (iii) in sterile corneal ulcers (corneal melts) in rabbits and humans [1012 ]. based on our identification of the site on the tetracycline molecule responsible for its mmp - inhibitory properties, that is, the zinc - binding -diketone moiety at carbons 11 and 12 [6, 7 ], we recently developed two new series of compounds that contain a similar zinc - binding site, but which are bicyclic rather than tetracyclic, namely, the chemically modified curcumins (cmcs) and the bis - aroylmethanes (bams). after testing the biologic activity of more than 30 of these novel compounds, using in vitro, cell, and tissue culture and in vivo rat and mouse models, we identified a compound, a phenylaminocarbonyl (triketonic rather than diketonic) curcumin, which showed the greatest efficacy in several models of diseases including diabetes, periodontitis, and osteoarthritis but demonstrated no evidence of toxicity when administered systemically in vivo either by oral intubation or by intraperitoneal injection or when tested in organ culture [1316 ]. in the current study, we demonstrate that this novel compound, cmc2.24, administered either topically or systemically, can substantially improve wound - healing in skin of the severely hyperglycemic type i diabetic rat. the details of the synthesis of cmc2.24 and its structure were described by us recently [14, 15 ]. the animal study was approved by the institutional animal care and use committee (iacuc) of stony brook university. in the first experiment, fifteen adult male sprague - dawley rats (body weight 300325 g, charles river laboratories international, inc., wilmington, ma) were injected through the tail vein with either 10 mm citrated saline buffer ph 4.5 (nondiabetic controls, ndcs) or the same solution containing streptozotocin (stz ; enzo life sciences, inc., plymouth meeting, pa ; 70 mg / kg body weight) to induce type i diabetes. the rats were then distributed into the five experimental groups described below (n = 3 rats / group). within 48 hours, the stz - injected rats exhibited severely elevated glucose levels in their urine. three weeks after inducing diabetes, the back skins of all the rats were shaved and a series of six standard wounds per rat, each 6 mm in diameter, were made using a surgical trephine. the following five experimental groups were established (in this initial experiment, treatment in all groups was for seven days ; a longer - term study is described below in experiment 3) : nondiabetic control (ndc) rats treated by daily topical application of white petrolatum jelly (vehicle) ; diabetic rats (d group) topically treated daily with vehicle alone ; diabetic rats treated by daily topical application of either a 1% (d + 1% 2.24) or a 3% (d + 3% 2.24) suspension of cmc2.24 in the vehicle ; and diabetics treated systemically by daily oral intubation of a 1 ml suspension of cmc2.24 in 2% carboxymethylcellulose at a dose of 30 mg / kg over the 7-day treatment protocol (d + 30 mg 2.24). collar (lomir biomedical inc., quebec, canada) was placed around the neck of each rat during the initial seven days of healing to prevent rats from inflicting self - injury, for example, biting and scratching, and to prevent licking of the wounds. at the end of this time period, the six circular wounds per rat were clinically assessed by measuring with a caliper the diameter of the wounds in millimeters, blood samples were collected, the rats were sacrificed, and skin samples were dissected for histological / histochemical and biochemical assessment as described below. the techniques described above are essentially the same as those described by us previously using topically or systemically administered tetracyclines (doxycycline and the chemically modified nonantimicrobial tetracyclines or cmts) in the diabetic male and in the surgically induced menopausal (ovariectomized) female, both recognized rat models of impaired wound - healing in skin [8, 9 ]. on day seven after creating the standardized wounds, all of the animals were anesthetized, blood samples were collected for blood glucose (one touch ultra glucometer ; johnson & johnson, new brunswick, nj) and hba1c (bayer a1cnow selfcheck, sunnyvale, ca) measurements, and, after the procedures below were completed, the rats were sacrificed by co2 inhalation. photographs were taken for clinical measurements to assess wound closure (18 wounds per experimental group). the percent reduction of the wound surface was calculated by measuring the diameter (in millimeters) of each wound before and after the treatment protocol. wound tissues on day 7 each pool of tissue was homogenized, extracted at 4c with 5 m urea in 50 mm tris - hcl buffer (ph 7.8) containing 0.2 m nacl and 5 mm cacl2 overnight, and then centrifuged for 1 hour at 11,000 g, as described by us previously. the supernatants were dialyzed against the tris - hcl, nacl, and cac12 buffer, and the proteinases were partially purified by ammonium sulfate added to 60% saturation. the precipitated proteinases were analyzed by elisa for collagenases mmp-8 (sigma - aldrich life sciences inc., st. louis, mo) and mmp-13 (tsz scientific llc, framingham, ma). biopsies of each of two wound sites, including surrounding nonwounded tissue, were taken and fixed in 10% neutral buffered formalin for 24 hours and then transferred to 50% ethanol prior to grossing, alcohol dehydration, xylene clearing, paraffin embedding, and sectioning. five - micron sections were stained with h&e and masson 's trichrome and the distance between wound margins was measured histomorphometrically using a calibrated ocular micrometer and confirmed image analysis [8, 9 ]. the last two wounds per rat were dissected, hydrolyzed, and analyzed for hydroxyproline as described below. in the second experiment, using the same duration (7 days) of treatment as described in the first, eighteen male sprague - dawley rats (300325 g body weight) were divided into six groups (n = 3 rats / group) ; some groups are different from those described in the first experiment i : nondiabetic control rats treated topically with vehicle alone (ndc) ; group ii : diabetics treated with vehicle only (d) ; groups iii and iv : diabetics treated topically with either 0.25% or 1% cmc2.24 suspended in white petrolatum jelly, once daily for seven days ; groups v and vi : diabetics treated topically with either 0.25% or 1% curcumin (sigma - aldrich, st. photographs were taken for clinical observations every day. at sacrifice, wound tissues and blood were collected for biochemical and histological analysis as described above. in the third experiment, a similar protocol was followed except that the twenty - four adult male rats were distributed into the following 6 experimental groups (n = 4 rats per group) : group i : ndc rats treated by daily application of white petrolatum jelly (vehicle) for 14 days ; group ii : d rats treated with vehicle (14 days) ; and group iii : d rats treated with 1% cmc2.24 for 14 days. the remaining twelve rats were distributed into the same three experimental groups ; however the topical treatment of the ndc and d groups, with either vehicle alone or 1% cmc2.24, was carried out for 30 days after 3-week duration of stz - induced diabetes. at the end of the 14-day and 30-day treatment protocols, the physical measurements and histologic and histochemical assessments were the same as those described above for the 7-day experiment. in addition, for all three time periods, tissue samples from the 6 mm punch biopsies were hydrolyzed twice in 2 n naoh at 120c for 1 hour each time. 50 l aliquots of the skin tissue hydrolysates were then analyzed for hydroxyproline, an amino acid essentially found only in collagen [17, 18 ]. all values are expressed as the mean the standard error of the mean (sem). as shown in figures 1(a) and 1(b), all four groups of d rats (including vehicle - only - treated d animals plus those d rats treated either topically or systemically with cmc2.24) exhibited significantly elevated blood glucose and hemoglobin a1c levels compared to the ndc rats, and none of the topical or systemic cmc2.24 treatments produced any detectable effect on the severity of hyperglycemia. however, despite this lack of benefit on blood glucose levels in the d rats, all of the cmc2.24 treatments (both topical and systemically administered) and, to a lesser extent, the curcumin treatments (figures 3 and 4) enhanced wound - healing in vivo, as indicated in figure 1(c) and as measured below. as expected, clinical appearance of the standardized skin wounds in these animals was consistent with impaired healing (figure 1(c)). note that, three weeks after inducing diabetes, the d rats were treated once each day, either topically or systemically, for an additional seven days with either cmc2.24 or vehicle alone (topical only) ; the ndc rats were only treated topically with vehicle alone. all of the d rats treated either topically with 1% or 3% cmc2.24, or systemically by oral gavage with 30 mg / kg of this compound, showed improvement and, in some groups, complete normalization of wound diameter was seen (figures 1(c) and 2). when the diameter of the wounds was measured using a caliper, the ndc rats, after 1 week of vehicle - alone treatment, showed a 30% reduction in wound size compared to the original 6 mm diameter lesion (figure 2). however, experimental diabetes reduced wound closure by 77% (p 0.05 ; not significant) than that seen in the ndc group. considering the other two treatments, orally administered cmc2.24 appeared to also normalize wound - healing in the d rats (d + 30 mg / kg versus ndc ; p = 0.66) but was not significantly more effective than 3% cmc2.24 topically administered (d + 30 mg 2.24 versus d + 3% 2.24 ; p = 0.09). the latter treatment regimen, although seemingly the least effective of the three, appeared to improve wound - healing by 228% compared to the vehicle - treated d rats (d versus d + 3% 2.24 ; p 0.05, 0.25% 2.24 versus 1% curcumin). two different collagenases, mmp-8 (collagenase-2) and mmp-13 (collagenase-3), were assessed in the wound tissues on day 7 using elisa techniques. in brief, mmp-13, which in rats is constitutive, analogous to mmp-1 in humans, did not show altered levels when comparing the ndcs to the various d groups (data not shown). in contrast, mmp-8 (also known as leukocyte - type collagenase) did show differences between the various groups of rats (figure 4). a tenfold increase in mmp-8 (p 0.05) on mmp-8 levels in the diabetic wounds, the 25% reduction of mmp-8 by 1% cmc2.24 was statistically significant (p = 0.005) (figure 4). the histologic assessment (masson 's trichrome staining) of wound - healing in the different groups of rats (figure 5) was generally consistent with the measurements described above. as shown in figure 6, 4/12 of the ndc wound biopsies assessed histologically showed complete reepithelialization at day 7 after creating the lesions, whereas diabetes appeared to reduce the incidence of reepithelialization by 50%. once again, two of the three cmc2.24 treatments, the 1% topically applied and the oral administration of 30 mg / kg body weight, essentially normalized this measure (i.e., reepithelialization) of wound - healing, whereas topical 3% 2.24 appeared slightly less effective. consistent with this pattern, the histomorphometric assessment of wound reepithelialization demonstrated that the d group was 27% less than ndc (p = 0.027) and, compared to the d group, the efficacy of the three different treatments was as follows : 1% 2.24 (p = 0.01) > 30 mg / kg 2.24 (p = 0.03) > 3% 2.24 (p > 0.05) ; only the latter treatment was not statistically effective (p = ns ; figure 6) compared to the vehicle - treated d group. the histologic measurement of wound diameter (mm) on day 7 showed a similar pattern of change : the 38% larger diameter of the diabetic wounds, compared to the wounds in the ndc rats, was normalized by both the 1% topical and the systemic treatments with cmc2.24, whereas the 3% cmc2.24 did not significantly reduce the histologic wound diameter (data not shown). based on the superior efficacy of the 1% cmc2.24 topical treatment, the longer - term effects of this regimen were also examined. figure 7 shows the progressive closure (% reduction of wound diameter) of the wounds in the different groups of rats at days 14 and 30, after creating the standardized lesions, compared to the day 7 data already described (see figure 2). the pattern of closure for the different experimental groups at day 14 was similar to that for day 7 ; that is, for day 14 (figure 7), the diabetic rats showed a 22% reduction in wound closure compared to the ndc rats (p 0.05 : ns). however, by day 30, all three experimental groups showed 100% reduction in the diameter of the standardized wounds (figure 7). the pattern was different for reepithelialization of the wounds in the three experimental groups (figure 8) ; that is, unlike the day 7 and day 14 data (see figure 7) which demonstrated significant impaired wound closure in the vehicle - treated d rats compared to the ndcs, and normalization of closure at these two time periods in the d rats treated with cmc2.24, as shown in figure 8, at days 14 and 30 all three groups (ndc, d, and d + 1% 2.24) showed complete (100%) reepithelialization of the standardized skin wounds assessed histologically. however the chronic status of the wounds in these various groups is not equivalent even after thirty days (when reepithelialization is complete) as is evident from the biochemical analysis of collagen content, now described. the data in figure 9 shows the collagen (assessed as hydroxyproline) levels in the wound tissues of the three experimental groups, ndc, d, and d + 1% 2.24 at days 7, 14, and 30 after creating the 6 mm diameter standardized lesions. in the nondiabetic control rats, the hydroxyproline levels in the healing wounds rapidly increased by over 500% (p = 0.008) by day 14 and remained at this level at day 30 the hydroxyproline levels in the vehicle - treated (d) and cmc - treated (d + 1% 2.24) diabetics increased much more slowly and did not reach the high levels in the ndc rats even at day 30. however, at this late time period, cmc2.24 treatment of the d rats wounds did increase the hydroxyproline levels by 140% (p = 0.003), compared to the vehicle - treated d rats, although the cmc2.24-treated diabetic wounds still showed hydroxyproline (collagen) levels 37% lower (p = 0.028) than those seen in the ndc rats on day 30. matrix metalloproteinases (mmps) are a multigene family of more than 25 zinc - dependent neutral proteinases involved in the degradation of extracellular matrix, nonmatrix constituents, and basement membranes. in addition, they process cytokines, chemokines, and growth factors, all of which participate in physiologic connective tissue turnover including bone remodeling and growth, embryonic morphogenesis, angiogenesis, and wound - healing [20, 21 ]. dysregulation of mmps is associated with a variety of inflammatory, apoptotic, and carcinogenic processes which mediate a substantial number of diseases such as (but not limited to) arthritis, atherosclerosis, periodontitis, and tumor invasion and metastasis [2224 ]. mmps play a role in all stages of wound - healing including inflammation, debridement of damaged connective tissue and reepithelialization in the early stages, and angiogenesis and connective tissue remodeling at later stages [25, 26 ]. impaired wound - healing is a well - known complication of diabetes which too often leads to chronic infection, amputation, and even death [3, 4 ], and virtually every aspect of wound - healing can be adversely affected by poorly controlled diabetes and hyperglycemia ranging from leukocyte dysfunctions and impaired reepithelialization to decreased fibroblast activity, suppressed collagen synthesis, and delayed regeneration. with regard to the mmps, several studies have shown that the induction of experimental diabetes increases mmp expression and activity in tissues such as skin and gingiva [9, 26, 27 ] and that administration of nonantimicrobial tetracyclines (tcs) can reduce this excessive proteinase activity down to essentially constitutive / physiologic levels seen in nondiabetic / normal rats. additional experiments demonstrated that both antimicrobial and nonantimicrobial tcs, applied either topically or systemically, normalized lesions in both diabetic and surgically induced menopausal rat models of impaired wound - healing [8, 9 ]. recent studies have applied this approach in clinical trials involving nonhealing wounds in diabetic patients [5, 25 ], and topical 1% doxycycline ointment healed the chronic diabetic ulcers better than the vehicle treatment. mechanisms could include the dampening of the pathologically excessive levels of collagenases (mmp-1 and mmp-8) and gelatinases (mmp-2 and mmp-9) that have been observed in biopsies of diabetic foot ulcers, compared to the levels of these proteinases seen in normal posttraumatic wounds. it should be recognized however, as previously discussed, that excessive inhibition of mmps can be deleterious since these enzymes have physiologic functions [21, 22 ]. as one example, constitutive levels of mmp-8 have anti - inflammatory properties, which is consistent with the observation that mmp-8 knockout mice exhibit increased inflammation and delayed wound - healing. in part, because of the limitations inherent in the use of tcs for wound - healing, including the potential for inducing the emergence of antibiotic - resistant bacteria and photosensitivity, we recently developed a new series of bicyclic rather than tetracyclic compounds with mmp - modulating activity associated, at least in part, with a similar -diketone, zinc and calcium - binding site in the tc molecule. initially, these bicyclic compounds included the use of the bis - aroylmethanes (bams) as well as a series of chemically modified curcumins (cmcs) [14, 15 ]. however, the initial lead compound, cmc2.5 (a triketonic 4-methoxycarbonyl curcumin), and, more recently, a newer and more potent compound, cmc2.24 (a phenylaminocarbonyl curcumin, also triketonic), showed superior efficacy in models of various collagenolytic diseases such as arthritis, periodontitis, and diabetes [1316 ]. the current report is the first to describe the efficacy of cmc2.24 in an animal model of impaired wound - healing, namely, the severely hyperglycemic diabetic rat. in brief, assessment of the healing of standardized 6 mm diameter wounds in the dorsal skin of the diabetic rats, using clinical, histologic / histochemical, and biochemical measures, all indicates that either topical or systemic (by the oral route) administration of cmc2.24 significantly enhanced wound - healing and that this cmc is substantially more effective than the parent molecule, curcumin. it should be noted that several studies have described wound - healing benefits using natural curcumin [30, 31 ]. it should be also noted that cmc2.24 did not appear to significantly affect wound - healing in the nondiabetic control rats (data not shown). various aspects of the wound - healing process were significantly improved, even normalized, in this animal model of severe type i diabetes even though none of the treatments with this compound appeared to reduce the severity of hyperglycemia. in the early stages, delayed reepithelialization of the diabetic wounds was returned to normal by either topical or systemically administered cmc2.24. a later event, connective tissue regeneration in the dermis, also appeared to be significantly improved, if not completely normalized, in the diabetic rat wounds during cmc2.24 treatment ; this beneficial effect was associated with increased collagen levels, assessed histochemically and by hydroxyproline analysis, and decreased pathologically excessive mmp activity. in this regard, mmp-8, which in the current study was generated in dramatically elevated levels during diabetes in the early inflammatory phase of wound - healing, was significantly reduced by cmc2.24 treatment ; it is important to note that both mmp-8 (collagenase-2) and mmp-9 (gelatinase b) are released when polymorphonuclear leukocytes degranulate during the inflammatory response. in this regard, preliminary data in our lab indicates that this gelatinase / type iv collagenase, like mmp-8, was also increased by diabetes and that cmc2.24 treatment suppresses this excessive mmp during wound - healing a similar pattern of change for this gelatinase was recently seen by us during experimental diabetes and its treatment with cmc2.24 in gingiva and skin. in contrast, mmp-13 (collagenase-3), which in rats appears to be constitutive and analogous to mmp-1 (collagenase-1) in humans [19, 33 ], did not appear to be affected either by diabetes or by cmc2.24 during the 7-day treatment of the wounds (data not shown) indicating that this novel treatment selectively inhibits pathologically excessive mmps but does not suppress constitutive mmps needed for normal / physiologic connective tissue turnover. mmp-14 also appears to play a role in wound repair and we have found that cmc2.24 is a potent inhibitor of this enzyme 's activity. however, whether cmc2.24 affects the expression of mmp-14 has not yet been determined. this study also demonstrated both the short - term and long - term benefits of topical cmc2.24 treatment in this model of impaired wound - healing. in this regard, cmc2.24 accelerated the reepithelialization and closure of the wounds in the severely hyperglycemic rats in the short term (7 and 14 days after wounding). however, in the long term (day 30), this treatment also increased the collagen content of the dermis, which in the vehicle - treated diabetics remained at abnormally low levels. the excess mmp activity in the diabetic rats could also explain, at least in part, not only the reduced collagen levels in the wounds but also its reduced solubility (at 4c) which is known to reflect excessive collagen cross - linking and aging [35, 36 ]. of interest, cmc2.24 treatment was recently found to prevent this diabetes - induced defect. future studies are necessary to determine whether the improved level and quality (i.e., solubility) of the collagen in cmc2.24-treated diabetic rats reflects decreased mmp degradation of newly synthesized poorly cross - linked collagen (the preferred substrate for these mmps), or an increase in synthesis of collagen (new collagen is characteristically more soluble and less cross - linked), or a combination of both. moreover, in a recent preliminary study, which has begun to assess the substantivity of this treatment, the improvements in wound - healing produced by the 2-week regimen of topical cmc2.24 were maintained for at least 2 more weeks after the treatment stopped, thus indicating that the severe diabetic state did not cause the deterioration of the 2.24-improved wounds after the topical treatment was terminated (data not shown). based on the current data, we propose the following working hypothesis : namely, this novel therapeutic compound resolves, rather than suppresses, the inflammatory phase of wound - healing, recognizing that inflammation is abnormally prolonged during the uncontrolled diabetic state in the absence of the therapeutic compound. in support of this hypothesis, recent preliminary studies in our lab on diabetic rats examined inflammatory cell functions in culture following in vivo treatment with cmc2.24. the results indicate that diabetes impairs the chemotactic activity of these cells, which leads to an impaired ability to combat infection and which contributes to prolonged inflammation. however, administration of this novel compound normalized both the numbers of these leukocytes in the inflammatory exudate and their chemotactic activity preventing the prolongation of an incompetent inflammatory response and promoting more rapid wound - healing. these findings and conclusions are consistent with recent observations that proresolution lipid mediators improve delayed healing in a mouse model of type 2 diabetes. impaired wound - healing in skin and other tissues is a major, sometimes fatal, complication of diabetes mellitus. the current study demonstrated that a 1% suspension (better than 0.25% or 3%) of a novel chemically modified curcumin, a phenylaminocarbonyl triketonic curcumin (cmc2.24), was more effective, topically applied to standardized wounds in the skin of stz - diabetic rats, than diketonic natural curcumin in accelerating reepithelialization and collagen replacement in the presence of unaffected severe hyperglycemia. systemically administered cmc2.24 (oral gavage, 30 mg / kg qd) showed similar efficacy as the topical formulation, based on clinical, histologic, and biochemical (hydroxyproline / collagen ; matrix metalloproteinases / mmps) evidence, assessed over time periods from 7 to 30 days. future clinical studies are needed to assess the potential of this novel compound in the management of impaired wound - healing in diabetic patients. | introduction. impaired wound - healing in diabetics can lead to life - threatening complications, such as limb amputation, associated in part with excessive matrix metalloproteinase- (mmp-) mediated degradation of collagen and other matrix constituents. in the current study, a novel triketonic chemically modified curcumin, cmc2.24, was tested for efficacy in healing of standardized skin wounds in streptozotocin - induced diabetic rats. initially, cmc2.24 was daily applied topically at 1% or 3% concentrations or administered systemically (oral intubation ; 30 mg / kg) ; controls received vehicle treatment only. over 7 days, the diabetics exhibited impaired wound closure, assessed by gross and histologic measurements, compared to the nondiabetic controls. all drug treatments significantly improved wound closure with efficacy ratings as follows : 1% 2.24 > systemic 2.24 > 3% 2.24 with no effect on the severe hyperglycemia. in subsequent experiments, 1% cmc2.24 normalized wound - healing in the diabetics, whereas 1% curcumin was no more effective than 0.25% cmc2.24, and the latter remained 34% worse than normal. mmp-8 was increased 10-fold in the diabetic wounds and topically applied 1% (but not 0.25%) cmc2.24 significantly reduced this excessive collagenase-2 ; mmp-13/collagenase-3 did not show significant changes. additional studies indicated efficacy of 1% cmc2.24 over more prolonged periods of time up to 30 days. |
regenerative medicine (regmed) is an interdisciplinary field of research and clinical applications focused on the repair, replacement or regeneration of cells, tissues or organs to restore impaired function resulting from any cause, including congenital defects, disease, trauma and ageing. it uses a combination of several converging technological approaches, both existing and newly emerging, that moves it beyond traditional transplantation and replacement therapies. these approaches may include, but are not limited to, the use of soluble molecules, gene therapy, stem and progenitor cell therapy, tissue engineering (te) and the reprogramming of cell and tissue types. tremendous research developments and advantages in the stem cell arena have shed new lights on future therapeutic possibilities and it seems logical that actively modulating or guiding repair processes holds the promise of regenerating damaged tissues and organs in the body by stimulating previously irreparable organs to heal themselves [36 ]. the prospect of transplanting in vitro grown organs and tissues has been widely investigated in the field of te. rapid success seen with early te results have stimulated scientists and clinicians to further pursue regenerative ideas when dealing with tissue failure or loss [818 ]. a brief review in the recent bibliography concerning advances in te and regmed would raise the impression that we are almost there, being able to produce tissue replacement off the shelf, in the near future for everyone in need [11, 14, 17, 1924 ]. interestingly enough, that was exactly the spirit some 10 years ago [25, 26 ]. it seems literally spoken that we are able to fabricate constructs that look like tissue, smell like tissue and taste like tissue but not some that also function equally like one. an ever perpetuating evolution is yet to bring the long awaited revolution in the health sector. stop tissue engineering and start engineering tissues. a brief historical tour of regeneration and regmed constitutes along with a critical analysis of the present, the filter through which we put together the puzzle of its future. the myth of the great titan prometheus, as introduced by hesiod in his theogony (8 century b.c.) provides an icon for regmed. when he stole fire, a symbol for civilization and technology, to give it to mankind he received a cruel punishment. jupiter had him chained to the carpathian mountains, where an eagle by the name of ethon would pick at his liver every day, which would in turn regenerate during the night (fig. the ancient greeks were aware of the regenerating capacity of liver, hence they named it (greek :) after, meaning to repair oneself. the myth of the titan prometheus provides an icon for regmed. prometheus was known as the benefactor of mankind for his desire to assist mortals and give them many beneficial gifts that helped them to survive and live prosperous lives. his duty as a god was to form man from water and earth, and in doing so, gave them each a gift of strength or speed, craftiness or wisdom, and many other benefactors that improved their ways of living. the most well - known gift of prometheus was the gift of fire which prometheus stole from zeus lightning bolts. prometheus defiance and betrayal provoked zeus to have him chained to mount caucasus, where an eagle ate daily from his ever - regenerating liver. as an immortal titan, he stated that animals in the early developmental stages have a higher potential for regeneration. he propagated that biological form originates from undifferentiated matter and clearly favoured what would later be described as epigenesis. in the second chapter of genesis, providing another example of epigenetic regeneration, the lord god built up into a woman the rib that he had taken from the man. several thousand years later, the first transplantation of a moor s leg in replacement of a patient s diseased lower extremity was attributed to the physician finally, data from a survey of more than 10,000 mummies from prehistoric peru demonstrated that cranial trepanation was a routine procedure as early as 3000 b.c. this procedure was sometimes followed by alloplastic reconstruction, as demonstrated by shells, gourds and silver or gold plates found next to trepanated skulls. hence, it is evident that mankind has been following the endeavour of regeneration since the prehistoric ages. the dream to master regeneration is one primordial ambition like flying or communicating over distances and man can not rest until he has mastered this quest too. l homme, was the first to defy a divine meaning in biological phenomena and stated that the function of the human body can be interpreted by means of its physical and chemical properties. however, it was not until the middle of the 18th century that the idea of an abstract motive power of biological organisms was dismissed by the general public and the scientific elite, when lavoisier (17431794) postulated that the chemical processes governing life could be reproduced and studied in the laboratory. during the same time naturalists were taking sides on the phenomena of generation and regeneration : pre - formationists supported that appendages to be regenerated and organisms to be born pre - existed as miniatures at the site of interest. so, at the base of a severed lizard tail, in their conception a miniature tail was preformed and waited to be in the sperm or in the ovum of the human there existed a miniature homunculus that grew into a newborn infant. this theory was in accordance with the christian beliefs of creation and prevailed until the middle of the 18th century. on the contrasting end, came the aristotelean thesis that undifferentiated matter was able to give rise to life. this theory had been actually named epigenesis by william harvey (15781657) in his work on the generation of animals grossly repeating on aristotle s works. abraham trembley (17101784) produced several publications on the regenerative phenomena on freshwater polyps. he managed to obtain a clone of 50 polyps from one organism that he had quartered. he performed sections at every conceivable plane, contradicting pre - formational beliefs of the time. the question was posed : if the animal soul was the organizing and unifying element of life, how could a newly regenerated form arise ? until the end of the 18th century philosophical and religious debate linked to the science of regeneration was set aside, and epigenesis gained acceptance with the eventual ascendancy of epigenetic and developmental embryology bringing about a revolution in natural sciences. once the religious and social burdens had fallen, the 19th century was marked by a rapid succession of developments. rudolf l. k. virchow stated the famous omnis cellula ex cellula and through microscopic observations he confirmed the cell theory and established the idea of cells being the elementary units of life able to replicate themselves by division [35, 36 ]. in 1867, hypothesis. he suggested that all of reparative cells taking part in the regeneration of wounds come from the bloodstream (and therefore from the bone marrow). finally, at the end of the 19th century, barth observed that upon autologous bone transplantation in hounds the vast majority of cells die and leave a scaffolding behind to be slowly repopulated by new host cells and an adequate neovascular network. in 1912, alexis carrel, a french scientist working at the rockefeller research institute in new york, started a culture from a small slice of heart muscle taken from a chick embryo. his belief that cells in culture were inherently immortal dominated the early 20th century until in the 1960s leonard hayflick and paul moorhead defied this dogma and proposed the hayflick limit, according to which differentiated cells in culture are not able to replicate more than 4060 times and are bound to display signs of senescence with successive passages. however, some mutated lines possessed the capacity to replicate themselves forever, if kept undifferentiated in culture. by 1967 leroy stevens had developed a strain of mice, with a high incidence of spontaneous testicular teratomas. these neoplasias displayed a characteristic mixture of different tissues lined up next to each other randomly. by the end of the 1960s it was established that they originated from germ cells that were able to give rise to a plethora of different tissues. so the concept of pluripotency of germinal cells was introduced and this tumour cell was named embryonal carcinoma stem cell (ec). research with ec stem cells expanded considerably in the 1970s. in a series of experiments, chimeric mice were produced by injecting ecs into early blastocysts. because ectopic blastocyst injections were also found to generate teratomas it became soon evident that pluripotent cells could also be derived from blastocysts directly. soon the next logical step was undertaken, when gail martin in the united states and martin evans in england generated in 1981 a stable diploid cell line that could generate every tissue of the adult body, including germ cells. researchers performed studies on cells obtained from blastocysts of primates, including rhesus monkeys and marmosets as well as human ec stem cell lines isolated from a rare tumour of the male testes, after orchiectomy procedures. however, in 1998 some couples undergoing treatment for extracorporeal fertilization donated a surplus of blastocysts for experimental purposes. james thomson isolated and cultivated a human es line from these blastocysts. a new era had dawned and the prospects of this technology seem overwhelming. in 1981, eugene bell from mit in boston drew some attention with his work on living skin equivalents. the year thereafter he received funding for preparing a cell - based vascular scaffold and in 1982 the funding companies claimed in a press conference to be pursuing research and development efforts in business segments including te and smart computer systems. a couple of years later, just down the aisle in mit, joseph vacanti of children s hospital approached robert langer with the idea of constructing custom made scaffolds for cell loading in an effort to overcome organ shortage for children in need of transplants. it was not until 1987 when during a special session of the us national science foundation meeting in washington dc the term the first conference on the engineering of living tissue was held in 1988 at lake tahoe, california and the proceedings of this meeting were published a year later as a book titled tissue engineering. in these proceedings a definition for the new field was given by robert nerem : tissue engineering is the application of the principles and methods of engineering and the life sciences towards the fundamental understanding of structure / function relationships in normal and pathological mammalian tissues and the development of biological substitutes to restore, maintain, or improve functions. this definition was cited again in brief in a 1993 publication by vacanti and langer in science maybe the most cited early review on te. two years later the bbc broadcast of images of a mouse from charles vacanti s laboratory with a tissue engineered ear auriculosaurus, was a turning point for te and aroused a huge public interest on new biotechnological advances. in 1996, the tissue engineering society international (tesi) was officially founded by joseph and charles vacanti, and the asian te societies were incorporated in tesi by 2000. soon thereafter, raymund e. horch and g. bjrn stark from freiburg encouraged the foundation of the european branch of tesi the etes, and they hosted in 2001 the tesi meeting in germany. hottest job for the future : with man - made skin already on the market and artificial cartilage not far behind, 25 years from now scientists expect to be pulling a pancreas out of a petri dish. by the turn of the century, there were over 3000 people working in the sector, with funding exceeding us $ 580 million [53, 54 ]. the transition from te into regmed was justified by dramatic developments in the financial, scientific and political landscape. from a financial point of view, the first years of the 21st century, anticipated to bring the biotechnological revolution, were characterized by a disconnect between expectations and reality. as lysaght noted in 2004 although aggregate development costs exceed $ 4.5 billion, the field has yet to produce a single profitable product. on the scientific arena new technologies had come into play, with the very term te being unable to accommodate them : the availability of human embryonic stem cells was bringing the cellular therapy at the front line of the field. gene technology had come to a point, where a whole mammal could be cloned or genetically manipulated (the monsanto swine case). the paramount significance of angiogenic processes [23, 24, 57 ] for homeostasis, biointegration and upscaling of tissue engineered constructs became clear [19, 5860 ]. experimental activities were directed to encompass integrative strategies towards generation of autonomously vascularized bioartificial tissue elements [61, 62 ] (fig. tumour research was further advanced with the prospect of revolutionizing cancer treatment to round up progress in the biotechnological arena [8, 26 ]. this is a replica of a neovascular network created by means of an arteriovenous loop in the rat model under influence of vegf(165). on the left side an overview demonstrating a relative nascent network (2 weeks after creation of the loop) with numerous new capillaries demonstrating different stages of arborization and remodelling. (c) neovascular sprouts (sprouting angiogenesis) are seen all around the picture, demonstrating a vivid ongoing angiogenesis. robert nicholas klein ii, a lawyer and real - estate developer, whose son suffered from diabetes mellitus type i, and whose mother suffered from alzheimer s invested over $ 3 million of his own money, and organized a public effort to put pressure on the government of california to change policy on stem cell research and regmed. on november 2, 2004, the proposition no. as a response to that, the california institute of regenerative medicine was established to supervise a huge funding of more than $ 3 billion over 10 years. the recent financial meltdown knocked the air out of biotech by devastating its already floundering inflow of investment funds. from the end of 2007 until 2010 the industry had no initial public offering introduction of shares in the public markets, and in the last 2 years we have witnessed a biotech asset yard sale with bigger pharma houses writing the cheque. the few survivors have turned their back to research altogether because like so often in science excellent scientific ideas and discoveries that provoke enthusiastic expectations turn out to be more difficult when it comes to routine clinical applications. investors are taking their money out of the industry thus driving stock price floors lower and by the end of 2009 as many as 79% of the biotech firms were trading under their opening price. no big hope for a cyclical recovery in funding exists momentarily because, as mentioned, investors have snubbed biotech for a decade. even at the peak of the nasdaq in october 2007 the percentage of industry players seeing their initial public enterprise value decline (trading under their opening price) was 59%. in the aggregate, even if the capital markets recover, it is unlikely that investors will flock to biotech hedge funds like it was believed earlier. vivid fantasies of lifesaving new cures and tantalizing financial forecasts in the 1990s have made space for tight fists and raised eyebrows among investors. however, this is hardly a phenomenon limited to the life sciences. since 1995 the gartner advisory group has devised a methodology to describe the ups and downs of new technologies and relate them to business considerations. these common general observations from the financial world seem to be also worthwhile to be considered by life science researchers, because of their analogy to scientific developments and their relevance to potential research funding sources. according to the hype cycles theory, a new technology is bound to undergo a series of five phases. as a first event there is a technology trigger. during this step, the new technology draws considerable attention by a breakthrough or a major press release. peak of inflated expectations during which over enthusiasm and overt publicity initiates a rage of investments. during this period there is a disconnection between expectations and performance. in the trough of disillusionment failure to meet the expectations, renders the technology unfashionable and unattractive in the public mind and for investors. during the slope of enlightenment although public interest has grossly deteriorated, in some centres the practical application and commercial potential is reassessed. finally, in the plateau of productivity the field re - emerges with viable concepts and profitability. understanding innovative technologies is pivotal to developing successful investment strategies in the biotech sector and hence is influential in developing the field of regmed. differences around the world seem to play a role. in a european conference held in may 2010 in brussels on innovation in healthcare with focus on small and medium enterprises (sme) relevant to the sector, there was a consensus that there are serious socio - economical barriers to be overcome, before the european landscape becomes nearly as favourable as in the united states. until then european grown talent will be constantly crossing the atlantic not only in respect to bioengineering expertise, but also in terms of talented fund managers. nevertheless, within the european framework programme (fp7) some 15% of a total of 6 billion dedicated to health research were funnelled into smes. another 2 billion has been separately invested in the innovative medicines initiative, a public private group that enhances collaboration between companies and experts on pre - competitive research projects. in autumn 2010 the european commission is about to publish the research and innovation strategy for the coming decade. the innovative medicines initiative represents an issue important to the ecosystem of health related research and development. although state funding is key to the foundling steps of innovative health technologies, eventually these advances will translate into products and services and will be integrated into the regular system of investment and revenue. there is a grey area, however, where the biotechnological advances escape the level of basic academic research but are also a step behind from becoming a proven concept ready for clinical application. this area has been described by several authors as the death valley and it is dictated not only by a financial gap but equally by a technical one as well as a gap of information and trust. specifically for small enterprises are unlikely to support a tedious effort of bringing an innovative technology to the point of applicability and profitability, whereas large - capital companies are better equipped, with a robust financial and political backbone. despite any financial up and downs and the driving forces of various funding sources regmed will definitely play a major role in the future of medical therapy. besides the ever growing knowledge of adult or embryonic stem cells and their regulation many patients might profit from new insights in the near future [6870 ]. as an example, preservation of cord blood stem cells is gaining popularity after promising results on their use for brain injury and type 1 diabetes mellitus as well as stroke and hearing loss. the capacity of liver to regenerate is known by millennia, but the dogma that heart is a nonregenerating organ has been only recently challenged. lately, niches of putative cardiac stem cells were identified in the murine and human heart [7678 ]. the necessity to discover the natural microenvironment and consequently, to engineer the artificial support essential for stem cells to act [7981 ]. finally, heart diseases might be prone to therapy by means of a new line of cells, the unique cardiac telocytes (fig. 4) responsible for a continuous regeneration in the mammalian heart [5, 10, 75, 82 ] and, possible repair, eventually. under the light of such recent achievements of research in regmed one might come back to the saga of prometheus to find out that it seems likely that not only the liver may carry the ability to regenerate in itself but also other vital organs like the heart might well be able to renew themselves. further molecular and cellular research into the functions of telocytes could well reveal new tools to regenerative reprogramming of lost organ functions. it is more than tempting to speculate that telocytes could be a novel, possible target for therapeutic strategies aimed at potentiating cardiac repair and regeneration after ischemic injury. digitally coloured transmission electron micrographs of mouse heart show telocytes (tc) in blue. (a) cardiac structural unit composed by cardiomyocyte (cm), blood vessel and tc. telocyte extends its characteristic, very long and thin, process (tp - telopode) between arteriole and cardiomyocyte. (b) regenerative cardiac unit composed by cardiomyocyte progenitor (cmp), capillary (cap), nerve and tc. many research projects in the field of regmed are dependent on state und industrial funding because public money is not available in sufficient amounts. the recent financial collapse has served as the straw that broke the camel s back in a decade - long erosion in the business fundamentals of the biotech industry. big pharmaceutical corporations have been acquiring some of the most significant biotech independents, possibly marking a new era where regmed leaves the academic labs of universities and the small boundaries of smes for gainful applications in the marketplace. the quest for regeneration is one of the primordial dreams of mankind, like flying, remote communication and setting foot on the moon. it is the destiny of mankind to reach for its visions, and therefore regmed is bound to succeed sooner or later. | abstractthe fields of tissue engineering (te) and regenerative medicine (regmed) are yet to bring about the anticipated therapeutic revolution. after two decades of extremely high expectations and often disappointing returns both in the medical as well as in the financial arena, this scientific field reflects the sense of a new era and suggests the feeling of making a fresh start although many scientists are probably seeking reorientation. much of research was industry driven, so that especially in the aftermath of the recent financial meltdown in the last 2 years we have witnessed a biotech asset yard sale. despite any monetary shortcomings, from a technological point of view there have been great leaps that are yet to find their way to the patient. regmed is definitely bound to play a major role in our life because it embodies one of the primordial dreams of mankind, such as : everlasting youth, flying, remote communication and setting foot on the moon. the journal of cellular and molecular medicine has been at the frontier of these developments in te and regmed from its beginning and reflects recent scientific advances in both fields. therefore this review tries to look at regmed through the keyhole of history which might just be like looking back to the future. |
radiation therapy is a key component of breast conservation therapy for in situ or invasive breast cancers. fisher 's landmark nsabp b-06 study showed at twenty - year follow - up a significant decrease in ipsilateral breast tumor recurrence from 39.2% to 14.3% (p < 0.001) when lumpectomy patients were treated postoperatively with whole breast irradiation. the early breast cancer trialists ' collaborative group 's meta - analysis of 7300 women treated with breast - conserving surgery and postoperative radiotherapy also showed a decrease in ipsilateral breast tumor recurrence over 5 years from 26% without radiotherapy to 7% with radiotherapy (p = 0.0002). in contrast to the nsabp trial, this study also showed a 15-year breast cancer mortality risk reduction from 35.9% without radiation to 30.5% with radiation (p = 0.005). therefore, the necessity of radiation in association with breast conservation surgery is widely accepted. radiation causes dna damage through the formation of free radicals which cause irreparable damage to cancer cells. browman and colleagues showed that head and neck cancer patients who continued to smoke during radiation therapy had lower rates of response and shorter disease free survival and overall survival than patients who did not smoke during radiation. however, this phenomenon has never been studied in patients undergoing radiation therapy for breast cancer. the purpose of this study was to compare the recurrence rates for patients who smoked or did not smoke during radiation therapy after breast conservation surgery. after institutional review board approval, the prospectively collected database at the breast center smilow cancer hospital at yale new haven was queried to capture cases of in situ and invasive breast cancers diagnosed between the years 2002 and 2010 treated with partial mastectomy followed by radiation therapy. patient demographics, tumor characteristics, further treatment including adjuvant systemic and radiation therapy, recurrence, and survival were obtained from both the database and the hospital tumor registry. patient charts were then reviewed to ascertain smoking status from the patient 's intake history form or from physician - documented histories or initial consultations. patient smoking history was categorized as never, former, current, or not available. former smokers were defined by the provider or patient through subjective history and the duration since last tobacco use was unknown. patients were treated in standard fashion with partial mastectomy and postoperative whole breast, external beam radiation therapy. treatment of the axillary field was at the discretion of the radiation oncologist and varied on a case - by - case basis depending on the burden of lymph node disease. the majority of patients were treated with standard 2-tangent whole breast irradiation if less than 4 lymph nodes were involved or 4 field, if more than 4 lymph nodes were involved. of 1166 charts reviewed on patients undergoing partial mastectomy, 624 had information regarding smoking status and follow - up documented and were included in this study. five of the included patients had follow - up at other institutions and were known to have had recurrence but it was not known whether the recurrence was local or distant. all cases of ductal intraepithelial neoplasia (din) were included, except for grade 1 din less than 2 mm in size, which is considered atypical ductal hyperplasia (adh). categorical data were compared using chi - square tests and kaplan - meier curves were constructed and compared with log rank (mantel - cox) tests for recurrence - free survival. of the 624 patients in the study, 52 (8.3%) were current smokers at the time of diagnosis, 196 (31.4%) were former smokers, and 376 (60.3%) were never smokers. smoking status was associated with race, patient age, and tumor stage, but not with grade, histology, or receptor status. african american women were significantly more likely to be current smokers than other racial / ethnic groups, and current smokers were significantly younger than nonsmokers. current smokers were more likely to have stage 0 disease (dcis) or stage 2 disease, whereas never smokers were most likely to have stage 1 disease. there was a trend for current smokers to have less infiltrating lobular cancer, but this was not statistically significant. there was no difference in estrogen receptor, progesterone receptor, or her2/neu receptor expression (or any molecular subtype combination such as er / pr, her2 +, or triple negative) by smoking history. at the time of analysis, the mean follow - up was 45 months and the median follow - up was 40 months (range 5130). there were 22 total recurrences ; the type of recurrence by smoking history is shown in table 2. there were too few cases to analyze the type of recurrence, but any recurrence was significantly more frequent in current smokers than in never smokers (p = 0.039). current smokers tended to recur sooner than never smokers, and prior smokers were intermediate between the two (p = 0.003 by log rank). multivariable cox regression models were constructed using race, age, stage, and smoking status and, as shown in table 3, both stage and smoking status remained significantly associated with recurrence. when adjusted for race, age, and tumor stage, current smokers were 6.7 times more likely to develop a recurrence than never smokers (95% ci : 2.022.4). there was no statistically significant difference, however, in overall survival by smoking status. this is the first study to show that, among breast cancer patients treated with partial mastectomy and radiation therapy, current smokers have a significantly higher recurrence rate than prior smokers or never smokers. although the numbers are small, almost 10% of current smokers had a documented recurrence at a mean follow - up of 45 months, compared with less than 4% for never and former smokers (2.7 and 3.6%, resp.). there was a tendency for current smokers to have more stage 2 disease whereas never smokers had more stage 1 disease. in a multivariable model, however, the key mechanism of action of radiation involves direct dna damage through formation of free radicals which cause irreparable damage, slowing the growth of cancers or causing direct cell death. as cancer cells are usually dividing at a faster rate than normal cells, they are more directly affected by radiation damage. when cancer cells are in a hypoxic milieu, they are thought to be less susceptible to radiation - induced damage. the results of the current study would seem to support the hypothesis of browman and colleagues who showed similar results for head and neck cancer patients who continued to smoke through radiation therapy. a review by holmes and colleagues of the nurses ' health study in 2007 found on multivariate analysis that current smokers had a 43% increase in adjusted relative risk of death from any cause. current and past smokers had higher rates of death from lung cancer, copd, and other lung diseases, but no direct increase in mortality from breast cancer. however, holmes ' study evaluated all methods of treatment for breast cancer in this diverse patient population of 5,056 patients. there was no significant difference in mortality among smokers compared with nonsmokers either with or without radiation. with regard to breast cancer and smoking status, prior studies have shown a decreased overall survival of smokers. calle and colleagues ' 1994 epidemiological study followed 676,530 female volunteers prospectively from 1982 to 1988 as part of the cancer prevention study ii through the american cancer society. during the study period, they found 880 deaths from breast cancer with an increased relative risk of death for current smokers (rr = 1.26 ; 95% ci : 1.051.50). furthermore, breast cancer mortality increased with total number of years smoked and number of cigarettes per day (p = 0.0096 and p = 0.0003, resp.). types of treatment, including surgery, drug therapy, and radiation therapy, were not included in this study.. followed 792 women diagnosed with breast cancer in sweden from 1977 to 1986. relative risk of mortality from breast cancer compared with never smokers was 1.44 for current smokers (95% ci : 1.01 to 2.06) and 1.13 (95% ci : 0.66 to 1.94) for prior smokers. all - cause mortality adjusted for age was 1.46 for smokers (95% ci : 1.15 to 1.86) compared to never smokers. data from 12,989 female patients in memorial sloan - kettering cancer center 's tumor registry was analyzed by yu and colleagues in 1997, and a history of smoking carried a risk ratio of 1.43 for all - cause mortality. this effect was seen not only for breast cancer, but also for oral and pancreatic cancers. it is interesting to speculate whether the results of the current study may have implications for breast cancer on a population level. a major epidemiologic problem has been to explain the increased mortality from breast cancer among african american women. most studies have focused either on reason for later stage of diagnosis or on biological factors leading to aggressive tumor behavior [10, 11 ]. however, the finding that african american women are more likely to be smokers, and that this may be detrimental, opens up a new avenue for investigation. prior epidemiologic studies have shown interesting molecular changes associated with african american race, smoking status, and breast cancer development. current smokers have also been shown to have increased rates of p53 mutations, independent of stage at diagnosis, which has been shown on multivariate analysis to be a predictor of poor prognosis. limitations of this study include the small number of current smokers and the uncertain location of recurrence in five patients as documented by the tumor registry. follow - up or smoking status was not available for 542 patients of the initial 1166 lumpectomy patients identified in our database, because the patient intake form was incomplete or because some patients only had consultations or part of their care provided at our center. in addition, it is unknown whether some patients may have stopped smoking after the diagnosis was made. future directions would include looking at a larger study population, examining postmastectomy radiation patients to see if they had similar outcomes and examining other concomitant epigenetic and environmental factors that may be confounding the data, such as bmi and socioeconomic status. if the results are really due to tissue hypoxia during radiation, one would expect this to primarily affect local and not distant recurrence. it is unclear at this time whether the results of this study should influence our treatment of breast cancer. although other factors could be confounding the findings, the results would suggest that smokers should be actively counseled to quit smoking at least through the duration of therapy. this could potentially even raise the question of not offering breast conservation therapy to active smokers, as they may be less likely to benefit from the effects of radiation therapy. | background. prior studies have shown earlier recurrence and decreased survival in patients with head and neck cancer who smoked while undergoing radiation therapy. the purpose of the current study was to determine whether smoking status at the time of partial mastectomy and radiation therapy for breast cancer affected recurrence or survival. method. a single institution retrospective chart review was performed to correlate smoking status with patient demographics, tumor characteristics, and outcomes for patients undergoing partial mastectomy and radiation therapy. results. there were 624 patients who underwent breast conservation surgery between 2002 and 2010 for whom smoking history and follow - up data were available. smoking status was associated with race, patient age, and tumor stage, but not with grade, histology, or receptor status. african american women were more likely to be current smokers (22% versus 7%, p < 0.001). with a mean follow - up of 45 months, recurrence was significantly higher in current smokers compared to former or never smokers (p = 0.039). in a multivariate model adjusted for race and tumor stage, recurrence among current smokers was 6.7 times that of never smokers (ci 2.022.4). conclusions. although the numbers are small, this study suggests that smoking may negatively influence recurrence rates after partial mastectomy and radiation therapy. a larger study is needed to confirm these observations. |
the tetraazamacrocyclic architecture of the tropocoronand ligand provides a flexible scaffold that can accommodate varied coordination environments and stabilize unusual or otherwise unachievable geometries at transition - metal centers (chart 1). in the absence of entatic tuning, cobalt(ii) metal centers display a proclivity toward high - spin octahedral or tetrahedral configurations, and cobalt(iii) centers prefer low - spin octahedral geometries. square - planar coordination environments are also readily observed for four - coordinate cobalt(iii) complexes, but small molecules with cobalt(iii) centers in tetrahedral environments are significantly scarce. the cobalt(iii) center in [cow12o40 ] exists in a tetrahedral environment, as does cobalt(iii) in [co(nor)4 ], where nor is the norbornyl anion. a small number of mononuclear cobalt(iii) imides have been prepared and structurally characterized, and in these species, the multiple - bond character of the metal the reason for the rarity of cobalt(iii) in tetrahedral environments becomes clear upon comparison of orbital splitting diagrams for a d metal center in square - planar versus tetrahedral geometries. the orbital filling diagram for a square - planar d center is shown in figure 1 for an s = 1 electronic configuration. the allocation of electrons corresponds to minimization of electron repulsion interactions and is the observed electronic distribution in [co(tc-4,4) ] complexes. in contrast, the orbital distribution in a d tetrahedral orbital splitting diagram results in the partial occupancy of two destabilized t2 molecular orbitals, with no contribution to minimization of electron repulsion interactions. that is, the transition from square - planar to tetrahedral geometry results in partial filling of orbitals that are destabilized in the tetrahedral configuration relative to the square - planar one, without minimizing electron repulsion. the square - planar configuration also minimizes the electron ligand repulsion from the dx y orbital. orbital correlation diagram for the transition from square - planar to tetrahedral geometry, shown for the electron occupancy corresponding to a d metal center. modified from albright, burdett, and whangbo. having the ability to control the environment at the metal center simply by changing the tropocoronand linker chain length places us in a unique situation to examine unusual coordination environments. the previously established series of four - coordinate cobalt(iii) tropocoronand complexes comprises [co(tc-3,3)](x) (x = bph4, bar4) and [co(tc-4,4)](bph4). these species exist in square - planar or distorted square - planar geometries, with twist angles, defined as the angle between planes formed by the metal and the two sets of aminotroponeiminate nitrogen atoms, of 8 and 41 for the four - coordinate complexes. in our pursuit to examine the size dependence of cobalt(ii) and cobalt(iii) tropocoronands on their ability to tune reactivity with nitric oxide, we prepared and structurally characterized two pseudotetrahedral cobalt(iii) complexes, [co(tc-5,5)](bf4) and [co(tc-6,6)](bph4). handling of air- and moisture - sensitive materials was conducted in an mbraun glovebox under a nitrogen atmosphere. methylene chloride and tetrahydrofuran (thf) solvents were purified by passage through activated alumina and stored over 4 molecular sieves under a nitrogen atmosphere prior to use. deuterated nmr solvents were obtained from cambridge isotope laboratories, stored under an inert nitrogen atmosphere, and used without further purification. the syntheses of [co(tc-5,5) ], [co(tc-6,6 ], [zn(tc-5,5) ], and [zn(tc-6,6) ] are described elsewhere. fc(bph4) was prepared according to previously published procedures. to a solution of [co(tc-5,5) ] (150 mg, 0.35 mmol) in methylene chloride was added fc(bf4) (94.4 mg, 0.346 mmol). the solution was evaporated to dryness and the resultant solid was washed with diethyl ether (et2o) to remove ferrocene. recrystallization from dichloromethane (dcm)/et2o at 30 c yielded x - ray - quality crystals (174 mg, 97% yield). uv vis nir [cdcl3 ;, nm (, m cm) ] : 349 (13650), 412 (15670), 757 (11420), 1166 (9183). calcd for c24h30bcof4n40.32ch2cl2 : c, 53.35 ; h, 5.64 ; n, 10.23. found : c, 53.33 ; h, 5.89 ; n, 10.21. evidence for ch2cl2 appeared in the nmr spectrum taken in cd2cl2. to a solution of [co(tc-6,6) ] (200 mg, 0.4 mmol) in methylene chloride was added fc(bph4) (218 mg, 0.434 mmol) in the dark. the solution was evaporated to dryness, and the resultant solid was washed with et2o to remove ferrocene. recrystallization from dcm / et2o at 30 c yielded x - ray - quality crystals (330 mg, 97% yield). note : we found methylene chloride solutions of fc(bph4) to be unstable and attribute variations in product yield to this property. nir [cdcl3 ;, nm (, m cm) ] : 271 (60900), 349 (21470), 424 (34560), 702 (2362), 981 (561), 1240 (475). calcd for c50h54bcon4 : c, 76.92 ; h, 6.97 ; n, 7.18. found : c, 76.68 ; h, 6.71 ; n, 7.33. to a solution of h2tc-5,5 (300 mg, 0.8 mmol) in thf was added nahmds (292 mg, 1.59 mmol), and the reaction was allowed to stir for 10 min. gacl3 (448 mg, 1.59 mmol) was added to the solution as a solid, and the reaction was left to stir overnight. the solution was evaporated to dryness, suspended in ch2cl2, and filtered through celite. recrystallization from ch2cl2/et2o at 30 c yielded x - ray - quality crystals (370 mg, 70% yield). h nmr (cd2cl2) : 1.68 (m, 8h, ch2), 1.91 (m, 4h, ch2), 3.74, (m, 4h, ch2), 3.85 (m, 4h, ch2), 7.10 (t, j = 10 hz, 2h, arh), 7.26 (d, j = 8 hz, 2h, arh), 7.64 (t, j = 12 hz, 2h, arh). esi - ms ([m gacl4 ]) : m / z 433.1 (calcd m / z 433.12). uv vis [cdcl3 ;, nm (, m cm) ] : 274 (sh, 63390), 280 (70810), 365 (40920), 416 (sh, 19480), 433 (27090). calcd for c24h30n4ga2cl4 : c, 43.96 ; h, 4.61 ; n, 8.54. found : c, 43.88 ; h, 4.44 ; n, 8.42. to a solution of h2tc-6,6 (54 mg, 0.13 mmol) in thf was added nahmds (45 mg, 0.27 mmol), and the reaction was allowed to stir for 15 min. gacl3 (70 mg, 0.27 mmol) was added to the solution as a solid, and the reaction was left to stir overnight. the solution was evaporated to dryness, suspended in ch2cl2, and filtered through celite. recrystallization from ch2cl2/et2o at 30 c yielded x - ray - quality crystals (52 mg, 57% yield). h nmr (cd2cl2) : 1.131.26 (m, 8h, ch2), 1.44 (m, 4h, ch2), 2.192.26 (m, 4h, ch2), 3.643.71 (m, 4h, ch2), 3.974.02 (m, 4h, ch2), 7.12 (t, j = 8 hz, 2h, arh), 7.35 (d, j = 8 hz, 2h, arh), 7.60 (t, j = 6 hz, 2h, arh). esi - ms ([m gacl4 ]) : m / z 471.1 (calcd m / z 471.20). vis [cdcl3 ;, nm (, m cm) ] : 272 (sh, 73750), 280 (83830), 363 (45720), 410 (sh, 22980), 422 (sh, 28930), 429 (37980). ir (kbr ; cm) : c = n 1514, calcd for c26h34n4ga2cl4 : c, 45.67 ; h, 5.01 ; n, 8.19. evan s method measurements were made in cd2cl2 on a 500 mhz varian inova spectrometer, and the temperature was measured by the residual peak separation of the h nmr of neat cd3od. optical spectra were recorded on a varian cary 5000 uv vis nir spectrophotometer in 6sq starna cells. fourier transform infrared spectra were recorded on a thermo nicolet avatar 360 spectrometer running the omnic software package. electrospray ionization mass spectrometry (esi - ms) analyses were performed on the agilent 1100 series lc / msd trap spectrometer. cyclic voltammograms were recorded under nitrogen using the versastat3 potentiostat (princeton applied research) and v3 studio software. a glassy carbon working electrode, samples were prepared as 35 mm solutions in methylene chloride with 0.1 m (n - bu4n)(pf6) as the supporting electrolyte. reported spectra the reversible fc / fc couple appeared at 0.57 v vs ag / ag. crystals were mounted in paratone n oil and frozen at 100 k under a cold nitrogen stream controlled by a cryopad low - temperature apparatus. data were collected on a bruker apex ccd x - ray diffractometer with graphite - monochromated mo k radiation (= 0.71073) controlled by the apex2 software package. the structure was solved by direct methods using shelxs-97 and refined by full - matrix least squares on f using the shelxl-97 program incorporated into the shelxtl software package. hydrogen atoms were assigned idealized positions and given thermal parameters 1.2 times the thermal parameters of the atoms to which they are attached. the structure of [ga(tc-6,6)](gacl4) contained voids filled with heavily disordered solvent molecules. the program squeeze(28) was used to remove the contributions of the disordered solvent to the structure factors. the electron density attributed to disordered solvent molecules created a channel along the 65 screw axis and corresponded to seven molecules of methylene chloride or et2o. the crystal of [ga(tc-6,6)](gacl4) was an inversion twin, and the percentage of the main twin component was refined to 58.2%. [co(tc-5,5) ] was successfully oxidized to [co(tc-5,5)](bf4) by reaction with ferrocenium tetrafluoroborate in dcm (figure 2, left, and tables s1 and s2 in the supporting information, si). structural characterization of [co(tc-5,5)](bf4) revealed the twist angle at cobalt to be 65. the average bond distance between the metal center and coordinating nitrogen atoms, co1nave, is 1.85, comparable to the 1.86 and 1.87 values in [co(tc-3,3)](bph4) and [co(tc-4,4)](bar4), respectively. thermal ellipsoid plots for [co(tc-5,5)](bf4) (left) and [co(tc-6,6)](bph4) (right), shown at 50% probability. we also oxidized [co(tc-6,6) ] to [co(tc-6,6)](bph4) with ferrocenium tetraphenylborate and structurally characterized the resulting cobalt(iii) product (figure 2, right, and figure s2 and tables s1 and s3 in the si). the twist angle in [co(tc-6,6) ] is 74, the largest value observed for cobalt(iii) tropocoronands to date. the co1nave bond distance is 1.82, somewhat shorter than that in previously reported cobalt(iii) tropocoronand coordination compounds. a comparison of bond lengths and twist angles for four - coordinate cobalt(ii) and -(iii) tropocoronand complexes is provided in table 1. tetrahedral geometry is rare for cobalt(iii) and, to our knowledge, has been observed in mononuclear small molecules only for [co(nor)4 ] and a handful of cobalt(iii) imido complexes to date. the paucity of cobalt(iii) in tetrahedral environments and the unusual geometries of the metal centers in [co(tc-5,5) ] and [co(tc-6,6) ] raised the possibility that oxidation of the parent cobalt(ii) compounds occurred at the ligand rather than the metal center. we therefore prepared and characterized [ga(tc-5,5)](gacl4) and [ga(tc-6,6)](gacl4) and compared their structural and electrochemical properties with those of the analogous cobalt complexes (figure 3 and tables s1, s4, and s5 in the si). thermal ellipsoid plots for [ga(tc-5,5)](gacl4) and [ga(tc-6,6)](gacl4), depicted at 50% probability. the [ga(tc-5,5)](gacl4) complex crystallizes in p21/c and exhibits crystallographic disorder over the entirety of the tropocoronand ligand (figure s1 in the si). the n4-coordinated gallium(iii) center has a twist angle of 81, displaying distorted tetrahedral geometry. the average gallium [ga(tc-6,6)](gacl4) crystallizes in p65 and exhibits nearly perfect tetrahedral coordination at the gallium(iii) center. the twist angle in [ga(tc-6,6) ] comparison of analogous bond distances between the [co(tc - n, n) ] and [ga(tc - n, n) ] complexes provided insight into the electron distribution in the former species (figures 4 and 5). nitrogen bonds in both cobalt complexes are shorter than those in the analogous gallium(iii) tropocoronands. n bond is probably a consequence of the greater covalent character of the cobalt tropocoronand compared to the gallium tropocoronand complex. comparison of structural parameters for [co(tc-5,5)](bf4) (left) and [ga(tc-5,5)](gacl4) (right). distances () shown are representative of bond distances within the tropocoronand ligand. comparison of structural parameters for [co(tc-6,6)](bph4) (left) and [ga(tc-6,6)](gacl4) (right). distances () shown are representative of bond distances within the tropocoronand ligand. in a review of the experimental and theoretical properties of transition - metal complexes bound to redox noninnocent ligands, ray. note difficulties in using x - ray structural parameters to draw conclusions regarding ligand- versus metal - based oxidation in delocalized systems, where the electron of a ligand radical is shared between two different ligands. the tropocoronand complexes provide such a delocalized system, for if a ligand radical were to form, the electron would be able to travel between the two aminotroponeiminate rings via the metal center. we are also aware of reports describing noninnocent ligands that undergo negligible structural rearrangement upon changes in redox state. we therefore turned to electrochemical methods for further evidence that oxidation of [co(tc-5,5) ] and [co(tc-6,6) ] results in cobalt(iii) species. previously published cyclic voltammetry studies of [co(tc-5,5) ] and [co(tc-6,6) ] were repeated and compared to the results of analogous studies of [ga(tc-5,5)](gacl4) and [ga(tc-6,6)](gacl4) (figure 6). we observed reversible couples at 0.344 v vs fc / fc for [co(tc-5,5) ] and 0.367 v vs fc / fc for [co(tc-6,6) ]. we assign these processes to metal - based redox reactions by comparison with electrochemical studies of [zn(tc-5,5) ] and [zn(tc-6,6) ]. the absence of similar reversible processes in the voltammograms of the zinc complexes is consistent with these redox events being metal - based. additionally, we assign the irreversible features at 0.467 v in the [co(tc-5,5) ] voltammogram and at 0.646 v in the [co(tc-6,6) ] voltammogram to ligand - based oxidations. the cobalt(ii)/cobalt(iii) couples observed here appeared at 100 mv more negative than those previously published. the reason for this discrepancy is unknown, and the current values are considered to be correct. cyclic voltammetry of [ga(tc-5,5)](gacl4) and [ga(tc-6,6)](gacl4) revealed ligand - based oxidations at 0.846 and 0.920 v vs fc / fc, respectively. ligand oxidation occurred at more positive potentials in the gallium(iii) complexes than in the analogous zinc(ii) and cobalt(iii) compounds. the shift to more positive potentials in the gallium(iii) tropocoronands relative to the zinc(ii) analogues can be attributed to the higher oxidation state of the gallium center. ligand oxidation in the gallium complexes may occur at more positive potentials than in the corresponding cobalt species because the cobalt center may be better able to stabilize the additional charge through covalent metal ligand interactions than gallium. cyclic voltammograms of (a) [co(tc-5,5) ], (b) [co(tc-6,6) ], (c) [ga(tc-5,5)](gacl4), and (d) [ga(tc-6,6)](gacl4), referenced to fc / fc. we attempted to characterize [co(tc-5,5)](bf4) by x - band electron paramagnetic resonance (epr) spectroscopy but were unable to observe any signal at 77 k. helium temperature epr spectroscopic studies of [co(tc-6,6)](bph4) were equally unrevealing. the h nmr spectrum of [co(tc-5,5)](bf4) shows broad, low - intensity peaks in the diamagnetic region of the spectrum, which may correspond to a tropocoronand - containing species (figure s3 in the si). peaks corresponding to protons of the tropocoronand ligand are absent in the h nmr spectrum of [co(tc-6,6)](bph4), but peaks for the tetraphenylborate counteranion are readily observed (figure s4 in the si). previous studies revealed that, as the total length of the linker chains (n + m) of a (tc - m, n) complex increases, both the geometry of the metal center and spin state change. the spin state of the distorted square - planar [ni(tc-4,5) ] is s = 0, and that of the distorted tetrahedral [ni(tc-5,5) ] is s = 1. in the cobalt(ii) system, a spin - state change is observed between [co(tc-4,4) ] (s = /2) and [co(tc-4,5) ] (s = /2). we previously reported that [co(tc-3,3)](bph4) (twist angle = 8) and [co(tc-4,4)](bph4) (twist angle = 41) have spin state s = 1 at room temperature and magnetic moments of eff = 3.1 and 3.6 b, respectively. to determine whether a spin - state change occurs in the [co(tc - m, n) ] series, the magnetic susceptibility of [co(tc-6,6)](bph4) was measured by evan s method at room temperature. the magnetic moment was determined to be 5.38 b, as expected for s = 2, confirming that a spin - state change does occur. calculations were performed with the use of orca(40) to supplement our understanding of the electronic structure of the [co(tc - n, n) ] series (n = 36). in all cases, the optimized geometries [bp / svp(tzvp on co) ] of the s = 2 state had twist angles 1321 larger than that those of the s = 1 state (table s6 in the si). this result supports the connection between the spin - state change and the square - planar to tetrahedral change in geometry. subsequent energy computations at the b3lyp / def2-tzvp level predicted that the s = 1 state is more stable than the s = 2 state in all four species, with triplet quintet gaps of 24.9, 18.9, 8.0, and 7.2 kcal / mol for n = 36, respectively (figure s6 in the si). our experimental results show that a spin - state change to s = 2 occurs for n = 5 or 6. the computations suggest that the ground - state electronic structure for n = 5 and 6 is s = 1, but there is a low energy barrier to accessing the s = 2 state (78 kcal / mol). even though this level of theory incorrectly predicts the ground state spin for n = 5 and 6, the trend in the tripletquintet energy gap across the series n = 36 is correct. quintet energy gap correlates almost linearly with the experimental twist angle (r = 0.936 ; figure s6 in the si). electronic transitions predicted for the series support the spin - state change but are less conclusive (figures s7s10 in the si). molecular orbitals involved in the electronic transitions for all complexes contained significant mixing between the cobalt and tropocoronand orbitals. thus, assignments for the transitions in the electronic spectra could not be made without further work. comparison of structural and electrochemical properties of the cobalt and gallium species [co(tc-5,5) ], [co(tc-6,6) ], [ga(tc-5,5) ], and [ga(tc-6,6) ] confirmed the cobalt(iii) character of the metal centers in [co(tc-5,5)](bf4) and [co(tc-6,6)](bph4). the synthesis of [co(tc-5,5)](bf4) and [co(tc-6,6)](bph4) augments the number of cobalt(iii) species having the rare pseudotetrahedral geometry. together with previous results from our laboratory, the present study reveals that a spin - state change from s = 1 to s = 2 occurs as the ligands tune the geometry of the complexes from pseudo - square - planar for [co(tc - n, n)](bph4) (n = 3, 4) to pseudotetrahedral for [co(tc-6,6)](bph4). | the preparation and characterization of two mononuclear cobalt(iii) tropocoronand complexes, [co(tc-5,5)](bf4) and [co(tc-6,6)](bph4), are reported. the cobalt(iii) centers exist in rare pseudotetrahedral conformations, with twist angles of 65 and 74 for the [co(tc-5,5]+ and [co(tc-6,6)]+ species, respectively. structural and electrochemical characteristics are compared with those of newly synthesized [ga(tc-5,5)](gacl4) and [ga(tc-6,6)](gacl4) analogues. the spin state of the pseudotetrahedral [co(tc-6,6)](bph4) compound was determined to be s = 2, a change in spin state from the value of s = 1 that occurs in the square - planar and distorted square - planar complexes, [co(tc-3,3)](x) (x = bph4, bar4) and [co(tc-4,4)](bph4), respectively. |
e. coli is a gram - negative bacterium of the enterobacteriaceae family, and is an important symbiotic member of the physiological flora of the large intestine of humans and warm - blooded animals. its basic functions in physiological conditions include decomposition of food and production of vitamins of b, k and c groups. it is isolated quite often from soil and water, where it comes from contaminations such as sewage or feces. there are many strains of these bacteria, differentiated based on somatic, superficial and ciliary antigens of various virulence based on adhesive abilities, capsule structure and production of toxins. thus, these microorganisms, beneficial in their natural environment, may suddenly become the cause of dangerous infectious diseases : diarrhoea, hemorrhagic enteritis, infections of the urinary tract, surgical wounds, nosocomial pneumonia, and finally, a fatal sepsis. early detection of a pathogen, especially a pathogen causing infectious disease of clinically significant manifestation and at an atypical site, may in some cases be essential for selection of appropriate, targeted antibiotic therapy enabling effective treatment or effective prophylaxis of the infection. the optimal diagnostic method, considering the biology of e. coli strains, would enable fast identification of bacterial cells in the biological sample (infected tissue, foodstuff, water) without the need to carry out laborious preparatory (incubation) procedures. unfortunately, current diagnostic methods do not meet basic requirements assuring efficacy of therapy, that is, the time required to obtain microbiological result. inoculation and phenotypic methods require up to several days of culture, whereas methods based on genetic identification (pcr) available only in a few centres, are very expensive and require specialized equipment. coli 0157 (merck) give results in as few as 20 minutes after placing a sample, but require its preparation for many hours (incubation). obviously, even the quickest test whose purpose is to detect a single specific pathogen, using a guided approach, can not determine the final clinical diagnosis of an infection, if only because of a potential risk of infection with mixed bacterial flora. to gain clinical significance, such a test should provide the pattern of sensitivity to medication of the detected microorganism. for several years we have been investigating identification of pathogenic factors of bacterial infections with the use of electrophoresis. although current technology allows only a relatively reliable identification of a single pathogen, such as escherichia coli, the construction of the apparatus allows us to combine techniques and to detect conditions, which in future may broaden the spectra of the specific pathogens responsible for mixed infections. we evaluated the diagnostic potential of a method of capillary zone electrophoresis according to methodology of buszewski for screening identification of pathogenic strains of escherichia coli. the above method was used for the first time in clinical conditions for instant microbiological evaluation of biological samples infected with e. coli. the study enrolled 60 consecutive patients of the clinic of general gastroenterologic and oncologic surgery of the collegium medicum in bydgoszcz, nicolaus copernicus university diagnosed with superficial infection of surgical wounds. the reference method for microbiological diagnosis of escherichia coli and other infections were classical, culture phenotypic studies carried out in the department of microbiology of regional hospital in torun. in 4 cases, verification was repeated due to suspicion of coexisting infection in the examined sample after the use of the cze method. the infected biological sample was taken from a surgical wound with symptoms of complications in the form of a superficial infection from trophic ulceration of similar character. it was usually a 0.51.0 ml sample of a secretion taken in aseptic conditions with a sterile syringe and placed in 1.5 ml of sterile water (aqua pro injection). when the density or amount of the secretion prevented simple sampling (aspirations to a syringe), the wound was rinsed with 1 ml of water, and then the sample was taken by the method above. the material, poured into sterile, tight, transport test - tubes, was immediately transported to the laboratory and introduced into cze apparatus directly from the transport container. [25 ]. at the same time, with the use of a microbiological spatula, a swab was taken from the wound and subjected to classical microbiological diagnostics. after obtaining the phenotypic result and confirming that only e. coli was present in the sample, in the same, sterile way bacterial cultures were taken from a petri dish and placed in 1.5 ml of sterile water (aqua pro injectione) and then subjected to the same electrophoretic analysis. clean, standard electropherograms characterizing the presence of individual pathogenic strains of analyzed bacteria were obtained by this method. each of the 60 sites from which microbiological test material was taken was catalogued. in 31 subjects these were infected postoperative wounds after resection of the large and the small intestine (group a), in 8 subjects they were trophic ulceration of the lower extremity due to chronic venous insufficiency or ischemia (group b), in 7 patients specimens were taken from infected wounds after urgent laparotomy in the course of peritonitis following perforation of the gastrointestinal tract (group c), in 10 subjects they were from suppurative wounds following other surgical procedures of the alimentary tract without affecting the intestinal continuity (eg, infected necrosis of the pancreas in acute pancreatitis) (group d) and in 4 subjects they were from foot ulceration in diabetic foot syndrome (group e). the system of measurement consisted of the following elements : system hp{ce (agilent technologies, waldbronn, germany) equipped with dad (diode array detector) with diode matrix technology enabling formation of the continuous spectrum of the examined substance in real time. the apparatus was connected with working station kayak with chemstation software (hewlett - packard) for operating the apparatus and collection of data. fused silica capillaries (composite metal services, worcester, uk). tbe buffer optimized towards e. coli consisted of 4.5 mm tris/4.5 mm, boric acid/0.1 mm edta (disodium ethyleneaminetetraacetate) of ph=8.53 diluted with deionized water in 8 : 1 ratio with addition of 0.2 g peo/40 ml of solution (polymer concentration 0.5%). the polymeric solution was then dispersed in an ultrasound bath for 4 hours at 60c. finally, dilution with tbe buffer to the concentration of 0.0125% completed preparation of the final buffer. the study enrolled 60 consecutive patients of the clinic of general gastroenterologic and oncologic surgery of the collegium medicum in bydgoszcz, nicolaus copernicus university diagnosed with superficial infection of surgical wounds. the reference method for microbiological diagnosis of escherichia coli and other infections were classical, culture phenotypic studies carried out in the department of microbiology of regional hospital in torun. in 4 cases, verification was repeated due to suspicion of coexisting infection in the examined sample after the use of the cze method. the infected biological sample was taken from a surgical wound with symptoms of complications in the form of a superficial infection from trophic ulceration of similar character. it was usually a 0.51.0 ml sample of a secretion taken in aseptic conditions with a sterile syringe and placed in 1.5 ml of sterile water (aqua pro injection). when the density or amount of the secretion prevented simple sampling (aspirations to a syringe), the wound was rinsed with 1 ml of water, and then the sample was taken by the method above. the material, poured into sterile, tight, transport test - tubes, was immediately transported to the laboratory and introduced into cze apparatus directly from the transport container. [25 ]. at the same time, with the use of a microbiological spatula, a swab was taken from the wound and subjected to classical microbiological diagnostics. after obtaining the phenotypic result and confirming that only e. coli was present in the sample, in the same, sterile way bacterial cultures were taken from a petri dish and placed in 1.5 ml of sterile water (aqua pro injectione) and then subjected to the same electrophoretic analysis. clean, standard electropherograms characterizing the presence of individual pathogenic strains of analyzed bacteria were obtained by this method. each of the 60 sites from which microbiological test material was taken was catalogued. in 31 subjects these were infected postoperative wounds after resection of the large and the small intestine (group a), in 8 subjects they were trophic ulceration of the lower extremity due to chronic venous insufficiency or ischemia (group b), in 7 patients specimens were taken from infected wounds after urgent laparotomy in the course of peritonitis following perforation of the gastrointestinal tract (group c), in 10 subjects they were from suppurative wounds following other surgical procedures of the alimentary tract without affecting the intestinal continuity (eg, infected necrosis of the pancreas in acute pancreatitis) (group d) and in 4 subjects they were from foot ulceration in diabetic foot syndrome (group e). during these tests, the system of measurement consisted of the following elements : system hp{ce (agilent technologies, waldbronn, germany) equipped with dad (diode array detector) with diode matrix technology enabling formation of the continuous spectrum of the examined substance in real time. the apparatus was connected with working station kayak with chemstation software (hewlett - packard) for operating the apparatus and collection of data. tbe buffer optimized towards e. coli consisted of 4.5 mm tris/4.5 mm, boric acid/0.1 mm edta (disodium ethyleneaminetetraacetate) of ph=8.53 diluted with deionized water in 8 : 1 ratio with addition of 0.2 g peo/40 ml of solution (polymer concentration 0.5%). the polymeric solution was then dispersed in an ultrasound bath for 4 hours at 60c. finally, dilution with tbe buffer to the concentration of 0.0125% completed preparation of the final buffer. of 60 examined, blind biological samples, isolated infection of e. coli was diagnosed in 30 by classical microbiological methods (in 19 subjects from group a, 1 subject from group b, 5 subjects from group c, 5 subjects from group d and no subject from group e), and those samples were selected for further analysis. complicated infections accompanying e. coli were diagnosed in 10 remaining cases (other bacterial species, fungi) and they were rejected from the examined group. in the final analyzed group of 30 infections with e. coli, in 26 cases pathogen was concomitantly diagnosed within as few as 45 minutes, based only on pathognomonic electropherogram of the sample. interestingly, in as many as in 3 of them, based on the character of the obtained cze diagram, a concomitant infection with cocci forming clusters was diagnosed with a high probability. on the following day classical inoculations from these 3 wounds were repeated. in the obtained, repeated results, concomitant infections with staphylococcus sp. strains (2) or enterococcus sp. all of these cases were also rejected from further processing for e. coli due to the risk of interaction of bacterial cells of various species under conditions of cze. a similar, but opposite situation was observed in a group of 20 infections with bacteria different from e. coli. in these 3 cases, coexistence of signal typical for e. coli was recognized by the cze method ; however, it was not confirmed primarily by a reference method, but in this case, subsequent verifying inoculations were not carried out. based on the final analyzed 52 (2 26) biological samples of isolated infections, a standard electropherogram that characterized presence of bacterial cells of escherichia coli was determined (figure 3). the typical peak characterizing presence of e. coli appeared as a single peak in time range from 2 to 5 minutes within flat - wide, complex and irregular signal (matrix - effect). such a single high signal in the electropherogram was considered a pathognomonic picture for e. coli infection, as well as for cases of complex infections (figures 3,4). matrix effect being an irregular, wide signal, contained in 015 minutes band, was a picture of sample background, which in this case consisted of solid particles and chemical substances of tissue excretion (eg, epithelial cells, morphotic elements of blood, fibrin, minerals) (figure 5). typical validation methods with respect to the finally selected uniform group of infections were used for determination of basic parameters (sensitivity and specificity) of the developed cze system as the diagnostic method for detecting presence of e. coli rods by screening. based on the described, simple comparative analysis, the initial degree of detection of e. coli infection was obtained in a biological sample of 86.7%. this sensitivity for cze set designed in this way was determined at the level of microorganism species. specificity of the method in the whole examined group reached 85.0% ; however, it should be considered that in the group of results considered as non - infected with e. coli, some cases could be found which were not detected primarily by phenotypic method (not verified by repeated inoculation). these parameters may indicate a high quality of the developed screening test already at this stage. morbidity of escherichia coli is strictly strain - dependent. there are both totally non - virulent strains that are important factors of intestinal homeostasis, and virulent strains causing various infectious diseases. in general, morbidity of these bacteria is revealed after occurrence of some favourable conditions, such as infancy, or a decrease of general and local immunity. in extreme cases, the infection may occur in the systemic form, with multi - organ failure, which is why e. coli is the most common factor causing generalized infections in newborns. there are 3 main groups of pathogenic e. coli : diarrhoea - causing strains epec, strains causing urinary tract infections upec, and strains causing sepsis sepec and cerebrospinal meningitis nemec. in natural conditions, e. coli is found in human and animal intestines as well as in soil and water, where it comes from secretion and feces. these bacteria may also colonize the skin and mucus membranes of the oral cavity and respiratory system. microbiological diagnostics of infections with these bacteria consists of classical inoculation methods and identification of strains with the use of agglutination techniques. diagnosis at the level of a strain is sometimes also made by highly specialized pcr methods. mean time to obtain a detection result by the traditional method can be from 2 to 7 days. treatment consists of using initially empirical and then targeted antibiotic therapy. starting the antibiotic therapy as soon as possible is very import in the treatment of most severe infections, such as when ensuring that the optimal choice of antibiotic in treatment of the detected bacteria species is based on empirical tables. sometimes, especially in newborns, hours determine life and death of a patient, but detection of the pathogen causing the disease may not be so quick. microbiological identification by traditional methods may be difficult many problems may be encountered that prolong the time to final result. complicated methods of sampling the material onto transport media and inappropriate conditions of transport to the microbiological laboratory may cause pre - laboratory errors that make results unreliable. identification analysis of isolated colonies, depending on selected method, may take, in case of e. coli, up to 24 hours. automation of determinations (eg, vitek 2) allows this time to be shortened to about 10 hours in the case of gram - negative bacteria, but a classical method of culture of microorganisms on petri dish before the test is still required. as few as 10 cells of o157 strain e. coli may cause infection, and such a low concentration may be insufficient for identification of a pathogen by currently used methods. in light of these factors, one of the most commonly investigated methods is combination of pcr and capillary electrophoresis, offering highly sensitive diagnostics for bacteria to be identified based on 16s rrna amplifications of individual bacterial strains. however, their drawback is time needed, requirement to employ additional specialists and very expensive and complicated laboratory equipment, including genetics and isotope laboratories. new diagnostic techniques such as capillary electrophoresis - based single - strand conformation polymorphism (ce - sscp) employ the relatively simple ce method, which significantly simplifies methodology without worsening parameters of the method. the method of microbiological determination based on electrophoresis process and pcr is in some cases widespread, such as during analyses of bacteriological purity of foodstuffs. this method is very sensitive and can be modified, for example by addition of multiplex pcr, in order to isolate electrophoretic signals of certain bacterial strains from a bacterial mixture even in a microscopic sample. chip technique is an innovation consisting of miniaturization of a pcr - based model and capillary electrophoresis, analyzing and counting the amplicones specific for certain bacterial strains obtained from just a few cells. such miniaturized equipment seems to be ideal for mobile microbiological identification, but it is still being developed and is not ready for commercial use. in our studies we decided to develop a very sensitive method that will meet the requirements of optimal microbiological screening diagnostics, by only slightly decreasing the specificity level with respect to the level of a strain. first stage testing should identify most common infections such as e. coli. as a principle, the method should be simple, fast and most important, possible to perform in the place of biological material sampling. calibration of the method for selected strains of e. coli showed that simple morphological analysis of the electrophoretic spectrum of the sample (eg, obtained as a result of ecg evaluation) is sufficient for its identification from a highly complex biological sample (eg, pus). earlier studies of buszewski on standard models of selected bacterial strains have confirmed that presence of electropherogram signals in their typical place (time to appear) and configuration (mutual order of many peaks) may be of pathognomonic value [35 ]. are equally valuable, and will be published next. after conducting the first clinical applications, it seems that all assumed characteristics of a good screening test in the cze method were obtained, but it still requires improvement. at its present stage, the used equipment enables placing it in any room and it only requires basic service. analysis does not require preparation of the sample, allowing it to be directly placed into a measuring apparatus immediately after being taken from the examined site. high speed the time required to identify escherichia coli cells in settled buffer solution tbe+peo did not exceed 30 minutes. compared with traditional methods identifying this species of bacteria, this is a crucial result, levelling the time to obtain it with routine, basic blood analyses. sensitivity and repeatability based on the analyzed group of patients with surgical wound infections, a 86.7% sensitivity was obtained, as well as in cases of mixtures of bacteria of various species. specificity of this method was calculated to be 85%. with maintenance of stable parameters of the study, moreover, in several cases it detected presence of other bacterial species colonies, thus exceeding sensitivity of routine culture (phenotypic) methods. the obtained initial results place the above method at the level of a good screening test. further studies, supported by analysis of greater and more microbiologically complex groups, most probably will allow improvement of the method. rapid general definition of a pathogen may in the future become a significant factor improving prognosis of most severely ill patients with virulent e. coli strains, such as newborns, in whom it is a common cause of bacteraemia and sepsis. early initiation of empirical antibiotic therapy based on identification of the bacterial species causing the disease will significantly improve the results of treatment, buying time to obtain antibiograms by traditional methods. in each medical centre dealing with treatment of infectious diseases, a detailed table presenting empirical sensitivity to antibiotics should be developed, based on the local and current microbiological statistics ; this is of particular importance since increasingly frequent infections with such common bacteria as e. coli are characterized by a wide spectrum of resistance to antibiotics. this method is limited at the present stage of development as a parallel and reliable identification of infections caused by mixed bacterial flora and evaluation of their sensitivity to medications is currently impossible. the present research, however, indicates that it is possible to combine tests oriented to other microorganisms, which may partly eliminate the above - mentioned weaknesses. this method may also be used as an industrial test for evaluation of contamination of food and water, with potential wide use in prophylaxis of bacterial infections, as well as in other, specialized epidemiological studies (eg, for a quick analysis of potential vectors of human infections with e. coli 0157 : h7, such as molluscs). it may be possible to improve the sensitivity of the method, reaching for virulence of individual strains, may be isolation with use of both superficial and intracellular cze - specific cell antigens obtained, for example, by homogenization. the classical cze method by itself clearly has great potential for detecting and identifying characteristic, species - specific, bacterial cells. these initial results of the clinical use of the method identifying presence of infection with e. coli based on electrophoretic signal raises hopes for creating a reliable, very fast and cheap screening test for use both in medicine and industry. | summarybackgroundescherichia coli is a gram - negative bacterium which is a basic, symbiotic element of the physiological flora of the large intestine of humans and warm - blooded animals. however, in specific cases it may become a very dangerous pathogen (eg, diarrhoea, infection of the urinary tract, lungs, and generalized infections). its early detection, as a cause of infectious disease, helps to achieve optimal treatment results ; however, classical microbiological tests require at least 24 hours from sample taking to diagnosis.material/methodswe present a unique solution based on cze technologies enabling identification of e. coli presence in studied sample within half an hour. altogether, 30 e. coli - infected wounds and ulcerations were examined, comparing the results obtained by classical culture method with the result of capillary zone electrophoresis (cze) electropherogram.resultsthe method, which does not require any preparation of the sample, achieved 86.7% sensitivity and 85%specificity in the examined clinical material (infections of surgical wounds).conclusionsthe obtained results enable reliable, very fast testing for e. coli as a pathogen. |
the safety of cerebral angiography has greatly evolved since its development by moniz, yet the prevalence of neurological complications is still 2.3%. estimated headache incidence after cerebral angiography is 0.3% although a 43% prevalence of migraine headache after angiography was described in migraine patients. one study designed to identify post - angiography headache used a small sample of cerebrovascular patients and reported 33% incidence of post - angiography headache. the international classification of headache disorders does not recognize post - angiography headache, only headaches starting during angiography or within 24 h of embolization. we aimed to study post - procedure headaches, in order to (1) establish its frequency, (2) identify predisposing factors to its occurrence and to (3) determine its relation to previous headaches. a prospective cross - sectional observational study was conducted on consecutive adult patients undergoing digital subtraction angiography (dsa) in a university hospital ; the study protocol was approved by the hospital s ethical committee. a semi - standardized interview was applied before the procedure, with demographic, clinical and technical data, screening, characterization and classification of previous headaches, angiographic findings and/or therapeutic results. one to 5 days after the procedure, an author blinded to previous history applied an identical interview to each patient and registered current clinical status ; the same evaluation was made 38 months later, by telephone. spss v11.5 was used ; frequency analysis performed with non - parametric tests, hypothesis on differences in means between groups tested by independent samples t test. the relation between post - procedure headaches and the risk factors found significant in bivariate analysis was studied with binary logistic regression ; the best fit chosen based on likehood ratio. a total of 107 patients (67 females, age 52 16.3) and 122 procedures were included ; only the first procedure of each patient was analyzed, summing 86 diagnostic dsas, 19 embolizations and two angioplasties studied. indications for dsa were subarachnoid hemorrhages (32 patients), stroke (20) and others (unruptured aneurisms, post - therapeutic controls, avms, arteriovenous fistulae, tumor). therapeutic procedures were embolizations of aneurysms (ten), avms (three), arteriovenous fistulas (four), tumor rammi (three) and carotid stenosis stenting (one). previous history of headache was reported by 47(43.9%) patients ; 23 with secondary headaches and 12 migraine. female gender was related to the presence of previous headaches (p = 0.005). seventy - eight (72.9%) patients were available for revaluation occurring on an average 28 15h 54 min after the procedure (mode 24 h) ; lost patients were early discharged (12) or had impaired communication (ete, aphasia, coma, confusion). forty (51.3%) patients reported headaches since the procedure17 continuous pain, 12 a single headache episode and 11 several episodes (average 4.4 5.5). a headache - free interval between the procedure and pain onset occurred in 31 patients, lasting 10.4 7.6 h. headache was a mild (59%) stabbing (45%) bilateral (55%) pain referred to the trigeminal territory (66.7%). most cases were without photophobia (61.5%), phonophobia (56.4%), nausea or vomiting (84.6%). headache was classified as migraine in three patients (one with previous migraine), tension - type headache (nine) and secondary in 28 patients. female gender (p = 0.013, rr 2.50, 95% ci 1.344.68) and diagnosis of aneurysm or sah (p = 0.022, rr 2.16, 95% ci 1.413.33) were risk factors for post - procedure headaches. separate group analysis of sah patients (n = 30) identified younger age (55.3 vs. 68.9, p = 0.034) and lower average contrast dye iodine concentration (327.6 vs. 357.1, p = 0.033) to relate to headache occurrence in these patients. in patients without sah (n = 48) only female gender (p = 0.005) presented as a risk factor, yet sample was too small to calculate relative risk. a telephonic evaluation performed 164 61.6 days post - procedure included 82 patients (63.4% females, age 51.3 16.8) ; patients lost to follow - up were due to impossibility of telephonic contact and death (one). forty patients (48.8%) described recurrent headaches since the procedure, 12 of which related its onset to the procedure. seven patients had less than a total of four attacks ; the remaining had 20.7 33.6 episodes monthly, ranging from one every 2 months to five daily attacks. the episodes lasted less than 12 h in 75% patients, average duration 2.7 5.2 h. six patients had daily persistent headaches. headaches were mild (62.5%) unilateral (60%) non - throbbing (76.9%) pain in the trigeminal territory without associated nausea / vomiting (72.5%) ; 50% had photophobia and effort intolerance, 60% phonophobia. headache was classified as migraine (12), tension - type headache (20) and secondary (8) with positive correlation (p = 0.008) to the same previous headache diagnosis. headache at 6 months was more likely in patients with previous headaches and migraine. patients, grouped by initial headache diagnosis and presence or absence of headache at reevaluations, are depicted in figs. 1 and 2.fig. 1previous headache diagnosis and headaches at each revaluation (24 h and 6 months) ; 1st ev (24 h) : evaluation 24 h after the procedure ; 2nd ev (6 m) : evaluation 6 months after the procedure ; x axis represents previous headache history ; numbers illustrated are patients counts within each groupfig. 2relation of headache diagnosis 6 months after the procedure to previous headache history ; x axis : headaches the 6 months revaluation ; h free : headache - free without headaches ; m : missing data ; z axis : recurrent headaches before the procedure ; p h free : previously headache free ; p secondary : previous secondary headache ; p tension - type : previous tension - type headache ; p migraine : previous migraine, m : missing ; y axis : number of patients in groups, as depicted in the gray - shaded legend previous headache diagnosis and headaches at each revaluation (24 h and 6 months) ; 1st ev (24 h) : evaluation 24 h after the procedure ; 2nd ev (6 m) : evaluation 6 months after the procedure ; x axis represents previous headache history ; numbers illustrated are patients counts within each group relation of headache diagnosis 6 months after the procedure to previous headache history ; x axis : headaches the 6 months revaluation ; h free : headache - free without headaches ; m : missing data ; z axis : recurrent headaches before the procedure ; p h free : previously headache free ; p secondary : previous secondary headache ; p tension - type : previous tension - type headache ; p migraine : previous migraine, m : missing ; y axis : number of patients in groups, as depicted in the gray - shaded legend headaches after angiography are common, yet information about them is insufficient its benign character is probably the reason why prospective series evaluating dsa complications fail to screen for headache. we report 35.4% incidence of headaches 24 h after angiography (excluding sah patients) in agreement with published data. we could not identify typical post - procedure headache characteristics, probably reflecting its multifactorial etiology. female gender was the only risk factor for post - procedure headache occurrence a higher prevalence of most headache types on females has been documented in epidemiological studies probably due to hormonal or pain perception threshold issues [79 ]. in opposition previous data, we did not find a relation neither with previous headaches nor migraine. post - procedure headache onset had a delay of hours after the procedure a fact previously reported [4, 5, 10 ] that has been attributed to a triggering effect of cerebral angiography in susceptible individuals. in our series, case reports of post - angiography migraine [1012 ] after dsa or embolizations for cerebrovascular disease, vascular malformations and cortical dysplasia [10, 12 ] were described. in four cases, intra - angiographic onset of vasospasm in the posterior brain regions occurred, then typical aura developed with subsequent headache ; one case had vasodilatation of the fronto - temporal meningeal vessels 24 h after the onset of a migraine - like headache without aura. it is possible that direct or indirect arterial stimulation induces the cortical spreading depression phenomena and consequent migraine - like headache, sometimes preceded by focal neurological symptoms localizing blood flow changes. as with our series, some of these patients did not have previous migraine. to our knowledge data suggests a relation to previous headaches (migraine in particular) so probably it is just a reflection of its recurrent pattern. patients often relate the onset / aggravation of complaints to procedures ; in our series, 19.6% of patients without previous headaches described new - onset headaches since the procedure, representing 22.5% of patients with recurrent headaches 6 months post - procedure. the explanation maybe a higher valorization of headache complains after an invasive intervention, also conditioned from increased self awareness and learning that headache can be a warning sign of intracranial pathology. in conclusion, early post - angiography headache is common and must be foreseen, especially in women ; it tends to be a mild headache starting a few hours after the procedure ; so, a simple analgesic scheme prescribed si opus sit can promote comfort. angiography does not seem to influence the occurrence of chronic recurrent headaches ; causality remains to be determined in the minority of patients whose chronic headaches develop after angiography. | headache is a common symptom after cerebral angiography, although it has seldom been studied. we aimed to evaluate the frequency of headache at 24 h and 6 months after angiography and to describe its characteristics. we used a cross - sectional survey of consecutive patients submitted to angiography and determined headache presence and its characterization. headache occurrence was analyzed against headache history, clinical data, technical and demographical variables. of 107 procedures studied, 51.3% patients experienced headaches within 24 h. patients more likely to experience headaches were females or had subarachnoid hemorrhage. six months post - procedure 48.8% of patients had frequent headaches. these patients had a positive headache history before the procedure, migraine in particular. half of patients undergoing routine angiography experience benign post - procedure headaches within 24 h (especially women), yet it does not seem to predispose to chronic long - term headaches. |
a total of 14 nicus from across the united states participated in this study, including tampa, florida ; wichita, kansas ; toledo, ohio ; salt lake city, utah ; birmingham, alabama ; cleveland, ohio ; allentown, pennsylvania ; san diego, california ; valhalla, new york ; manhasset, new york ; portland, oregon ; cleveland, ohio ; south bend, india ; and brooklyn, new york. the study population consisted of preterm infants born at 33 weeks gestational age with birth weights ranging from 700 to 1500 g who were enterally fed hm in the nicu. infants identified as eligible for randomization and for whom consent was obtained were randomly assigned to one of the 2 study regimens. sealed envelopes containing the subject treatment group assignment were prepared from randomization schedules that were computer - generated using a pseudorandom permuted blocks algorithm. a separate computer - generated randomization schedule was produced for twins to ensure that eligible twins were both assigned to the same product. the randomization was block stratified by birth weight (7001000 g and 10001500 g) and sex. eligibility criteria included appropriate intrauterine growth and maternal intent to provide breast milk during the study. the use of donor hm was not permitted during the study period unless indicated by the clinical staff or pi but could have been used in the first week of life before study initiation. infants were excluded for enteral feeds not started within 21 days of life, severe congenital anomalies, expectant transfer to another facility, 5-minute apgar 18 g kg day(7). the mean hc gain for both groups also closely matched recent recommendations for a hc gain of > 0.9 cm / wk (7). this result was not surprising given the excellent weight, length, and hc gains previously reported in infants fed pi - hmf powder (13). ehrenkranz (7) have reported that as the rate of weight gain increased in hospitalized preterm infants, the incidence of cerebral palsy, neurodevelopmental impairment, and need for re - hospitalization decreased significantly. a weight gain rate of > 18 g kg day and a hc growth rate of > 0.9 cm / wk were associated with better neurodevelopmental and growth outcomes. weight and length differed between the groups. although there were no significant differences in mean weight at birth or sday 1, infants receiving le - hmf had lb greater mean weight than the infants in the pi - hmf group at the end of the study period. although the rate of linear growth was not statistically different, infants in the le - hmf group had greater achieved linear growth during the study period. it is possible that the greater weight and length in the le - hmf infants was because of the higher number of infants in this group that adhered to the assigned study feeding. new expert recommendations suggest that extremely - low - birth - weight infants (< 1000 g birth weight) have higher protein requirements (3.54.5 g/100 kcal) (16). hmfs provide an important strategy to overcoming nutrient deficits for preterm and low - birth - weight infants. differences in the level and ingredient sources of the macronutrients, especially the protein quantity, in pi - hmf versus le - hmf may have contributed to the overall performance of the le - hmf group. the higher protein intake in infants receiving le - hmf (3.6 g/100 kcal) as compared to pi - hmf (3.0 g/100 kcal) was likely one of the reasons for the improved growth observed in these infants. although infants in the le - hmf group had higher protein intakes, energy intakes were not different between the groups. preterm infants fed fortified hm have variable rates of growth at least partly because of differences in intake of calories, carbohydrates, electrolytes, calcium, phosphate, and protein. the acid - base status of the preterm infant also, however, affects growth. in preterm infants the kidney may not tolerate an acid load, leading to the development of metabolic acidosis. in a recent study, a liquid acidified hmf caused metabolic acidosis and poor growth in preterm infants in the nicu (17,18). in another study, rochow (19) described a commercially available fortifier in europe that had to be reformulated because of the development of metabolic acidosis from an imbalance of electrolytes. the authors recorded a mean weight gain of only 9.7 g kg day and decreased bone mineralization with metabolic acidosis. the le - hmf protein source may be beneficial for this population because it was extensively hydrolyzed casein formulation without any intact cow's - milk protein. it has been suggested that a combination of free amino acids and short chain peptides (di- and tri peptides) may allow more optimal nitrogen absorption (20,21). intact bovine protein powder hmf has an excellent safety record ; however, a recent study by sullivan (11) suggested the possibility that even in the presence of a hm base diet, the addition of intact bovine protein powder hmf is associated with higher rates of total and surgical nec. although this study was not powdered for nec there was no difference in the nec or sepsis rates between the infants fed an intact bovine protein and the extensively hydrolyzed protein. intact bovine protein has higher associated long - term risk for allergy and atopy compared with hm - fed infants. because preterm infants have a similar risk for allergy and atopy compared with term infants and in the nicu have presented with symptoms suggestive of allergic colitis, avoiding intact bovine protein may be a desirable objective. for preterm infants fed hm the use of an extensively hydrolyzed protein - based hmf is an appropriate option. in general, blood chemistries were within normal reference ranges for preterm infants. the higher bun and prealbumin seen in the le - hmf group can be attributed to the higher protein content of le - hmf. it should be noted that although bun is influenced by renal function and hydration state, all other influences being equal, it is proportional to protein intake and responds rapidly to changes in protein intake (4,5,26,27). nearly 25 years ago kashyap showed that even a small deficit in protein intake impairs both growth in lean body mass and linear growth (28). in recent years, arslanoglu reported that addition of protein to preterm feedings of recovering vlbw infants resulted in significantly improved linear growth (4,5). this was accomplished by monitoring the bun level so that when it was less than 9 mg / dl, increased protein was added to their feedings. it was observed in the present study that the mean bun level fell < 9 mg / dl by week 2 in infants receiving pi - hmf ; however, in infants receiving le - hmf it never fell < 9 mg / dl during the entire study period. our results, in part, agree with other investigators that an increased protein - to - calorie ratio in the feeds of preterm infants will improve linear growth (4,5,9,28). it is becoming increasingly evident that promoting catch - up growth in the nicu may have implications for long - term development and health (7,29). the study examined the combined effects of changing both protein content and type (hydrolyzed vs intact). a number of subjects in this study did not complete the protocol to sday 29. this partially diluted the effects seen in the itt groups but still permitted demonstration of differential effects seen in the spf subgroup. infants < 700 g birth weight were excluded from this study and therefore the study findings can not be readily extrapolated to this vulnerable group. it is expected however that this group would have higher protein demands than infants in this study and therefore would be as likely or more to have a favorable response to higher protein. although no differences were seen between both groups for nec and sepsis the study size was too small to discern true differences for these outcomes. both fortifiers showed excellent tolerance and a low rate of morbidity outcomes, with the infants who were spfs fed le - hmf having improved growth. these data confirm the safety and suitability of this new concentrated liquid hmf for preterm infants. | abstractobjectives : this study was a comparison of growth and tolerance in premature infants fed either standard powdered human milk fortifier (hmf) or a newly formulated concentrated liquid that contained extensively hydrolyzed protein.methods:this was an unblinded randomized controlled multicenter noninferiority study on preterm infants receiving human milk (hm) supplemented with 2 randomly assigned hmfs, either concentrated liquid hmf containing extensively hydrolyzed protein (le - hmf) or a powdered intact protein hmf (pi - hmf) as the control. the study population consisted of preterm infants 33 weeks who were enterally fed hm. infants were studied from the first day of hm fortification until day 29 or hospital discharge, whichever came first.results:a total of 147 preterm infants were enrolled. noninferiority was observed in weight gain reported in the intent - to - treat (itt) analysis was 18.2 and 17.5 g kg1 day1 for the le - hmf and pi - hmf groups, respectively. in an a priori defined subgroup of strict protocol followers (n = 75), the infants fed le - hmf achieved greater weight over time than those fed pi - hmf (p = 0.036). the le - hmf group achieved greater linear growth over time compared to the pi - hmf (p = 0.029). the protein intake from fortified hm was significantly higher in the le - hmf group compared with the pi - hmf group (3.9 vs 3.3 g kg1 day1, p 18 g kg1 day1. |
a significant minority of women with newly diagnosed ovarian cancer are unfit for radical surgery or the outcome of multidisciplinary review is that surgery is unlikely to adequately debulk their extensive tumour. for this group, neoadjuvant chemotherapy is increasingly being used as the primary treatment with the aim of proceeding to subsequent surgery should the patient and/or disease bulk improve sufficiently to allow this. this protocol of neoadjuvant chemotherapy followed by interval debulking surgery (ids) was described in an eortc study reported in 1995. that approach is being further investigated in the eortc 55971 study and the mrc uk chorus (chemotherapy or upfront surgery) study. these studies address the timing of surgery relative to chemotherapy for newly diagnosed ovarian cancer and women believed to be suitable for surgery at diagnosis were randomised to receive either surgery followed by chemotherapy or neoadjuvant therapy followed by ids. early results from the eortc 55971 study indicate similar outcomes for the 2 randomised arms but lower surgical morbidity in the ids arm. the chorus study is continuing to recruit and an a priori plan is for meta - analysis of the 2 studies. the management implications of these studies are significant : at first sight the pivotal role of surgery in initial management is challenged. there has been much debate and some criticism of the study findings and recommendations but it seems clear that use of neoadjuvant chemotherapy is a safe alternative in this group of patients and does not compromise the standard of care. there are also significant implications for imaging and investigation of these women with suspected ovarian cancer. embarking upon neoadjuvant chemotherapy demands a confident histological diagnosis and this can be provided by image - guided core biopsy (igcb). this becomes more important as investigation of chemotherapy regimens specific to the different subtypes of ovarian cancer are being established. put simply, a diagnosis of adenocarcinoma based on cytological evaluation of ascitic fluid is increasingly insufficient. other cytological techniques such as preparation of a cell block specimen are untested in this regard. one tantalising question for future investigation is whether women could be treated by chemotherapy alone. in the ids protocol, women are treated with 3 cycles of chemotherapy, re - evaluated with computed tomography (ct), undergo surgery and then complete their chemotherapy with 3 or more further cycles of treatment. some women have such an impressive response to the initial 3 cycles of therapy that no residual disease is evident on the re - evaluation ct. the question has often been asked in our own multidisciplinary team meetings (mdtm) as to the purpose of removal of the apparently normal gynaecological apparatus and omentum at that stage. the surgery potentially interrupts the intensity of chemotherapy and/or causes morbidity in a woman who is starting to feel well again. the more recent joint eortc 55955/mrc uk ov05 study addresses another key question : what is the value of treating recurrent ovarian cancer diagnosed by ca-125 assay versus treating women who relapse clinically ? it has been known for many years that ca-125 levels increase weeks or months before there is any clinical or ct scan evidence of disease. in the above protocol, women with treated ovarian cancer who were in complete remission were followed up with clinical review including ca-125 measurements. neither the doctor nor the patient saw the results until they became abnormal. at that point the patients were randomised to be informed and treated with rechallenge chemotherapy or not to be informed and for chemotherapy to be withheld until there was clinical or imaging evidence of recurrent disease. the results of this study were recently presented at the plenary session at the 2009 asco meeting : the women treated in the 2 arms had no difference in survival but the patients in the ca-125 triggered arm had cumulatively received more cycles of chemotherapy, were treated about 5 months earlier for both second and third line therapies, and appeared to have worse quality of life score as judged by time to deterioration following randomisation. it has stated that women can be reassured that : there is no benefit from early detection of relapse by routine ca-125 measurements, andeven if ca-125 increases, chemotherapy can be safely delayed until there are symptoms or signs of tumour recurrence. mrc has recommended that women should be offered an informed choice in follow - up either to have no routine ca-125 measurements but rapid access to ca-125 testing if there are symptoms or signs of relapse or to have regular ca-125 measurements as is currently the case. there is no benefit from early detection of relapse by routine ca-125 measurements, and even if ca-125 increases, chemotherapy can be safely delayed until there are symptoms or signs of tumour recurrence. imaging has had no place in routine follow - up of women with treated ovarian cancer but is used responsively to an increased or increasing ca-125 level or to investigate symptoms suggestive of relapse. it now seems inappropriate to image any woman simply on the basis of ca-125 data. a variety of strategies have been suggested for the ct negative ca-125 positive woman ranging from recommencing chemotherapy to seeking imaging or other confirmation of the relapse. interval reassessment with ct, after say 3 months, is one option for the asymptomatic woman but alternatives tested include positron emission tomography (pet) or pet - ct. several studies have demonstrated the superiority of pet - ct over conventional diagnostic ct for the diagnosis of suspected ovarian cancer recurrence but its greatest utility is in the setting of the patients with increasing ca-125 levels and negative conventional imaging. the eortc 55971/ov05 study results suggest that use of these expensive and still scarce resources may not be justified for the asymptomatic woman. there is one small group of women with ovarian cancer for whom use of ct in follow - up had been thought appropriate. about 1 in 10 women are marker negative (mn) at diagnosis, i.e. they do not have increased ca-125 levels. our own policy has been to offer these women the option of ct follow - up but the ov05 results suggest that in future we might safely rely upon symptoms to trigger reinvestigation. in summary, the results of these collaborative studies from eortc and mrc uk are likely to have a significant effect on the management of ovarian cancer. ct evidence of recurrent tumour may become the standard of proof required for treatment of recurrent disease. | abstractfor many years the primary management of newly diagnosed advanced ovarian cancer has been cytoreductive surgery followed by chemotherapy, and the mainstay of follow - up of treated women has been serial assay of serum ca-125. the findings of 2 recent research protocols have a significant effect on these aspects of management. |
stereoacuity tests can be carried out easily and quickly to detect strabismus and amblyopia and to judge the degree of binocular vision after refractive correction [15 ]. some patients who are diagnosed as having no stereopsis by conventional stereoacuity tests, such as the titmus stereo test and tno stereo test, can enjoy three - dimensional (3d) movies. there are many differences between stereoacuity test devices used in clinical ophthalmology and movies and attractions that use 3d technology, such as whether they are static or dynamic, the target size, and the test distance. in the past, it was reported that a dynamic stereo target was more easily recognizable than a static stereo target [68 ]. devices also differ in test distance, target size, and binocular separation method [915 ]. recently, not only the near stereoacuity test, but also the far stereoacuity test has been used widely in clinical ophthalmology [1618 ]. there are many reports demonstrating that the far stereoacuity test is superior for detection of an abnormality (especially intermittent exotropia) versus the near stereoacuity test [17, 1924 ]. in addition, there is a report that stereoacuity, which the near stereoacuity test can not detect, is detected by the far stereoacuity test. to date, many studies on the effect of test distance on stereoacuity have been performed. however, in previous studies, there are various opinions about the effect of test distance on stereoacuity as a far stereoacuity test was easier to recognize than near one, there was no difference between far and near stereoacuity test [2630 ], and it depended on the subjects [3133 ]. however, the binocular separation method was not consistent, and target size did not necessarily correlate with test distance in previous studies. to investigate test distance, it is necessary to consider the target size, as target size decreases as the test distance increases. we investigated the effects of target size and test distance on stereoacuity using a 3d monitor that can display targets under various conditions, and we achieved our purpose. twenty - four subjects (mean age standard deviation, 21.8 0.8 years) participated in the study. no subject had ophthalmic disease other than minor refractive error, and each eye had distance and near vision values of 0.08 (logmar) under full refractive correction. far and near eye position of all subjects were less than 10. if the subjects felt fatigued during the procedure, the experiment was stopped immediately. this research conformed to the tenets of the declaration of helsinki and was approved by the kitasato university human sciences ethics committee (2010 - 020). potential subjects gave written consent after being given detailed information about the study and their role as a participant. informed consent was obtained from all subjects after an explanation of the nature and possible consequences of the study. we carried out stereoacuity tests on the subjects using the 3d visual function trainer orte (japan focus company, japan). the size of 3d visual function trainer orte is 24 inches (518.4 (width) 324 (height) mm) with a resolution of 1920 1200 pixels. we developed original software programs to display stereo targets. a polarization method (circular) this equipment was also used for both eyes in an open visual acuity test ; crosstalk is prevented, and the subject does not perceive the leakage of images (monocular cues are excluded). a 3d monitor showing the overall appearance and targets is shown in figure 1. analysis of variance (anova) and scheff 's method were used for statistical analysis ; p < 0.05 was considered statistically significant. for this examination, the test distance was 2.5 m and the target sizes were 0.1, 0.2, 0.5, and 0.9. the target shape was a circle, and its color was black. the thicknesses of the outlines of the circles at 0.1, 0.2, 0.5, and 0.9 were 1 mm, 2 mm, 4 mm, and 8 mm, respectively ; the inner gap sizes were 3 mm, 6 mm, 12 mm, and 24 mm, respectively. the room illuminance was 320 lx, and the luminance of the display was 400 cd / m. the amount of parallax presented using a 3d monitor depends on the distance between each pixel. therefore, 22 seconds was the minimum parallax that could be presented at a test distance of 2.5 m. we asked the subjects to choose which of the four targets was the stereo target. we asked the subjects to answer starting from the greatest amount of parallax (198 s) in descending order (an answer was judged correct if it was correctly answered all three times) ; when they responded with an incorrect answer, we judged the prior parallax as the stereoacuity. the test distances for this examination were 2.5, 5.0, and 7.5 m. we designed the experiment so that the target size doubles or triples when the test distance doubles or triples. therefore, the target sizes were 22, 44, and 66 mm ; the retinal target size was met at all test distances. the target for the visual angle at all test distance was 0.5. the thicknesses of the outlines of the circles at 2.5 m, 5.0 m, and 7.5 m were 4.4 mm, 8.8 mm, and 13.2 mm, respectively ; inner gap sizes were 13.2 mm, 26.4 mm, and 39.6 mm, respectively. the distances between the targets were 5.5 cm, 11.0 cm, and 16.5 cm. room luminance was 320 lx, and the luminance of the display was 400 cd / m. we asked the subjects to choose which of the four targets was the stereo target. we asked the subjects to answer starting from the greatest amount of parallax (198 s) in descending order (an answer was judged correct if it was correctly answered all three times) ; when they responded with an incorrect answer, we judged the prior parallax as the stereoacuity. the average stereoacuity values at target sizes of 0.1, 0.2, 0.5, and 0.9 were 59.58 14.86, 47.66 13.71, 41.25 15.95, and 39.41 15.52 seconds, respectively. anova was used in order to analyze the effect of the target size in stereoacuity. the effect of the target size was significant f(3,69) = 21.246, p < 0.0001. stereoacuity at a target size of 0.1 was significantly worse than those at target sizes of 0.2, 0.5, and 0.9 (p = 0.03, p < 0.0001, and p < 0.0001, resp.). average stereoacuity values at test distances of 2.5, 5.0, and 7.5 m were 44.91 16.16, 34.83 10.84, and 24.75 7.27 seconds, respectively. anova was used in order to analyze the effect of the test distance in stereoacuity. the effect of the test distance was significant f(2,46) = 29.295, p < 0.0001. stereoacuity at a test distance of 7.5 m was significantly better than at test distances of 2.5 and 5.0 m (p < 0.0001 and p = 0.02, resp.). stereoacuity at a distance of 5.0 m was significantly better than at 2.5 m (p = 0.04). our results showed that stereoacuity was significantly worse when the target size was 0.1. however, overall we observed a trend with stereoacuity becoming worse as the target size decreased. this can be explained by reduced visibility when the target size was smaller, therefore, resulting in a decrease in stereoacuity. we believe that the reason in cases where the retinal target size and presented parallax were the same is that when the test distance was increased, the projection rate of the stereo target (projection amount (distance of convergence point) from the 3d monitor / test distance 100) increased. in this experiment, for example, when the test distances were 2.5, 5.0, and 7.5 m and the presented parallax was 22 seconds, the projection amounts of the stereo targets from the 3d monitor were calculated as shown in figure 4 for subjects with a pupillary distance of 65 mm. in the case of a 2.5 m test distance, the distance between the right eye target and left eye target is 0.269 mm. the projection amount of a stereo target from a 3d monitor is calculated by using the following equation : 0.269 : 65 = x : (2500 x), x = 10.30 mm. in the case of a 5.0 m test distance, the distance between the right eye target and left eye target is 0.538 mm (0.269 2). the projection amount of the stereo target from the 3d monitor is calculated by using the following equation : 0.538 : 65 = x : (5000 x), x = 41.04 mm. in the case of a 7.5 m test distance, the distance between the right eye target and left eye target is 0.807 mm (0.269 3). the projection amount of the stereo target from the 3d monitor is calculated by using the following equation : 0.807 : 65 = x : (7500 x), x = 91.97 mm. the projection rates of the stereo target from the 3d monitor (projection amount of the stereo target from the 3d monitor / test distance 100) were 0.41%, 0.82%, and 1.23%, respectively. as the test distance increased, the projection rates of the stereo target from the 3d monitor increased, and the stereo target could be recognized more easily. if stereoacuity can be estimated, we can be confident that other binocular functions are good. parallax is currently used as an evaluation axis of stereoacuity. however, in different stereoacuity test conditions, parallax may be the same [3538 ]. our results show that the ease of determining stereoacuity was different under various conditions of target size and test distance. stereoacuity should be estimated not only by parallax, but also by other elements, including test distance and target size. | target size and test distance effects on stereoacuity were investigated in 24 subjects using a three - dimensional monitor. examination 1 : target size effects. the test distance was 2.5 m for 0.1, 0.2, 0.5, and 0.9 target sizes ; crossed parallax was presented in 22-second units. average stereoacuity values for 0.1, 0.2, 0.5, and 0.9 target sizes were 59.58 14.86, 47.66 13.71, 41.25 15.95, and 39.41 15.52 seconds, respectively. stereoacuity was significantly worse with a 0.1 target than with 0.2, 0.5, and 0.9 target sizes (p = 0.03, p < 0.0001, and p < 0.0001, resp.). examination 2 : test distance effects. test distances of 2.5, 5.0, and 7.5 m were investigated for a 0.5 target size ; crossed parallax was presented in 22-second units. average stereoacuity values at 2.5 m, 5.0 m, and 7.5 m test distances were 44.91 16.16, 34.83 10.84, and 24.75 7.27 seconds, respectively. stereoacuity at a 7.5 m distance was significantly better than at distances of 2.5 m and 5.0 m (p < 0.0001 and p = 0.02, resp.). stereoacuity at a 5.0 m distance was significantly better than at 2.5 m (p = 0.04). stereoacuity should be estimated by both parallax and other elements, including test distance and target size. |
with an estimated number of about 3000 species, distributed from central and south america, across africa to madagascar and southern india, cichlid fishes (cichlidae) represent the most species - rich family of vertebrates, accounting for about 10% of today 's teleost diversity [1, 2 ]. throughout their distribution range cichlids have repeatedly demonstrated their capacity of forming adaptive radiations explosive speciation with niche partitioning (reviewed in), generating an outstanding variation of body shapes, colour patterns and behaviour, and an enormous diversity of trophic and ecological specializations [46 ], which attracted numerous evolutionary biologists and established them as one of the prime model systems in evolutionary biology (e.g., [79 ]), but the greatest diversity of cichlid fishes is found in the east african great lakes. although endemic cichlid species assemblages are known from most east african lakes, the largest lakes tanganyika, malawi, and victoria harbour a particularly rich fauna of cichlid fishes, with an estimated number of 250800 species in each lake [1, 2 ]. thus, the number of species seems to be correlated with lake size, congruent with the expectation that species diversity increases with habitat heterogeneity and with the opportunity for isolation by distances and allopatric diversification. because of high degrees of endemism (9599%), these cichlid radiations most likely originated via intralacustrine speciation [1, 7, 10 ]. with an estimated age of 912 myr, lake tanganyika is by far the oldest of these lakes, and thus harbours the morphologically, behaviourally, ecologically, and genetically most diverse species assemblage [1, 2, 1215 ], although the number of species is the smallest. currently 200 valid species are recognized with several more awaiting scientific descriptions such that the total number of lake tanganyika 's cichlid species has been estimated to 250. these 200 species have been classified into 12 or alternatively 16 tribes, largely supported by molecular data. the specialized, diverse morphologies of these fish appear to be the result of adaptations in their respective niches, and thus, these fish are regarded as ideal model system for the study of adaptive radiation [4, 8, 9 ]. schluter defined adaptive radiation as the differentiation of a single ancestor into an array of species that inhabit a variety of environments and that differ in traits used to exploit those environments, and employed four features as criteria to detect adaptive radiation : (1) common ancestry, (2) phenotype - environment correlation (empirical evidence of correlation between the diverse phenotypes of descendant species and their divergent environments), (3) trait utility (experimental or theoretical tests of performance or fitness of a trait in its corresponding environment), and (4) rapid speciation. the first and fourth criteria are the subject of phylogenetic analyses of the species assemblage. the second and third criteria are to find adaptive phenotypes that differ between species as a result of divergent natural selection. these adaptive phenotypes may have caused reproductive isolation between species as byproduct (ecological speciation, e.g., [20, 21 ]) or allowed co - occurrence of two or more closely related species at the same place and in the same time (resource partitioning, e.g., [22, 23 ]). within the lake tanganyika numerous morphological studies on lake tanganyika cichlid fish have been published in the past. in this short review, we summarize the findings from these studies and highlight their importance for understanding the process of adaptive radiation. the first molecular phylogeny of lake tanganyika cichlids was published by nishida in 1991. this allozyme - based phylogeny resolved the relationships of 20 species representing all lake tanganyika cichlid tribes and suggested that the lake tanganyika cichlids were polyphyletic. subsequently, many molecular phylogenetic studies have been published, such that, in contrast to the situation in the much younger cichlid species flocks of lakes malawi and victoria, we now have rather precise knowledge on the phylogenetic relationships within the lake tanganyika cichlid species flock and the relationship among african lacustrine and riverine cichlid faunas (figure 1) (reviewed by [810 ]). with the exception of tylochromis polylepis and oreochromis tanganicae, the lake tanganyika cichlids evolved from a common ancestor after the formation of the lake 912 ma [24, 27, 33 ]. these fish are thought to have rapidly radiated within the lake, which fulfils the first and fourth criteria of adaptive radiation presented by schluter (common ancestry and rapid speciation). tylochromis polylepis and o. tanganicae colonized the lake only recently, thus establishing themselves in an already mature adaptive radiation [25, 31 ]. excluding these species, the lake tanganyika cichlid species flock comprises at least six major lineages. the substrate - brooding tribe lamprologini consists of about 80 species endemic to the lake, plus eight species that colonized the congo river and one species that colonized the malagarazi river after the intralacustrine radiation of this tribe [37, 38 ]. the mouth - brooding c - lineage (sensu) includes about 100 endemic species assigned to six or ten tribes (species numbers, phylogenetic relationships, and biological characteristics of tribes are reviewed in). whereas the monophyly of each tribe is well supported, the phylogenetic relationships among the tribes are still largely unresolved, indicating rapid diversification and adaptation to particular ecological niches at the onset of the lake tanganyika radiation. the tropheini, one of the endemic mouth - brooding tribes, were shown to be nested within the haplochromines, the most species - rich lineage that also includes the species flocks of the remaining east african great lakes and the majority of the northern, eastern, and southern african riverine cichlid species but originated in the course of the primary tanganyika radiation [29, 30 ]. the simultaneous radiation of the lamprologini and the c - lineage was probably triggered by the onset of deep - water conditions in lake tanganyika about 56 ma (primary lacustrine radiation) [27, 30, 38 ]. alternative hypotheses [39, 40 ] suggest a two- to fivefold older age for the lake tanganyika radiation which would considerably predate the establishment of a real lacustrine habitat [11, 41, 42 ] and imply that the onset of the radiation has happened in a riverine environment, a habitat generally considered as not suitable to host a radiation. the biological characteristics of the lake tanganyika cichlid species assemblage with a clear resource partitioning between most tribes and the relative age of the east african cichlid species flocks strongly argue for the lake tanganyika radiation to have happened in a single deep - water lake [10, 30 ]. unlike for the younger lakes malawi and victoria molecular phylogenies of lake tanganyika cichlids are typically well resolved, with most species, genera, and tribes being resolved as monophyletic, indicating that lineage sorting has been largely completed. thus, it has been assumed that these molecular phylogenies (typically based on mitochondrial genes) do closely approach the true species trees although the placement of some taxa was inconsistent with taxonomy [4446 ]. recent evidence from complementary analyses of nuclear dna, however, clearly demonstrated that, despite being well resolved, the mitochondrial phylogenies do not necessarily reflect the true phylogenetic relationships but might be severely misleading due to ancient incomplete lineage sorting, ancient (and recent) introgression, and even hybrid speciation [26, 38, 4753 ]. nevertheless, mitochondrial phylogenies have been and are still used as proxies of species trees in comparative approaches to study the interaction and evolution of biological traits in a phylogenetic context (e.g., [5457 ]), thus potentially introducing an error in inferred evolutionary patterns. the evolutionary success of cichlids has been attributed to the interaction of extrinsic environmental factors and intrinsic species - specific traits. some of these intrinsic traits might be naturally selected (e.g., the trophic morphology, body size, body shape, and visual pigments), whereas others are predominantly sexually selected (e.g., body colouration, smell, and courtship sounds), though distinctions between naturally and sexually selected traits might be not that clear (reviewed in). these studies may be classified into four major groups : studies of trophic morphology, body shape, body size, and nervous system. cichlid fishes exhibit a functionally decoupled set of jaws, the oral jaws and the pharyngeal jaws (figure 2). in particular the pharyngeal jaw is considered a key innovation, representing a key factor for the emergence of a diversity unparalleled among vertebrates [5961 ]. the pharyngeal jaw apparatus of cichlid fish is a functionally integrated and highly specialized system and considered to represent a major adaptive complex. decoupled from the oral jaws, the pharyngeal jaws are used for efficient crushing and processing of food items. thus, the oral jaws are freed from their dual task of food collection and preparation. due to this division of functions the development of numerous specializations of food collection and procession mechanisms became possible and minor modifications in oral and pharyngeal jaw structure allow for the utilization of novel food resources within a few generations, such that unexploited ecological niches can be rapidly occupied [62, 63 ]. thus, the specialized pharyngeal jaws in cichlid fish seem to be particularly important for their propensity to rapidly adapt to novel ecological niches, but other trophic morphological features have probably also played key roles for their rapid diversification. likely, rapid differentiation in trophic specializations results in effective resource partitioning and thus drives the evolution of complex cichlid communities by ecological segregation (e.g., [64, 65 ]). several studies on lake tanganyika cichlids described correlations of the trophic morphology with food habits (oral jaws and teeth [6569 ], pharyngeal jaws [59, 69, 70 ], preorbital region [15, 71 ], and intestine [72, 73 ]), suggesting resource - based divergent selection as an important diversifying force in lake tanganyika cichlids. in algae feeding species, for example, the shape of jaw teeth differs according to the resources they exploit. thus, the species of the genus petrochromis have tricuspid teeth in high density that make a brash - like structure to comb unicellular algae from filamentous algae on rocks, whereas species of tropheus and some other genera have large bicuspid teeth in the most anterior row that allow to nip and tear off filamentous algae from rocks. tooth shape both on oral and pharyngeal jaws in cichlid fish was shown to respond quickly to selection and change even within the lifetime of a single individual [74, 75 ]. recently, significant advances regarding the developmental pathways and genetic basis leading to different tooth shapes and numbers have been made [7678 ]. further work on this issue will certainly be important to fully understand how inter- and intraspecific differences in tooth shape and numbers emerge as a sometimes quick response to environmental / trophic constraints in species - rich adaptive radiations where species typically occupy a rather narrow ecological niche. besides the number and shape of teeth, the shape of the pharyngeal jaws and the oral jaws (or the preorbital region in general) are well known to strongly correlate with diet and were shown to respond quickly to natural selection when new habitats are colonized or when it comes to optimize feeding performance throughout ontogeny. thus, piscivorous fish were shown to have longer oral jaws than insectivorous and herbivorous fishes, and likewise, slender and elongated pharyngeal jaws have been found to be highly correlated with piscivory [79, 80 ]. however, recent evidence on the lake tanganyika cichlid lepidiolamprologus elongatus suggests that the mature piscivorous morphotype is refined by a relative widening of the caudal part of the lower pharyngeal jaw, which has been interpreted as prerequisite to the insertion of well - developed musculature and the construction of a powerful lever system which allows for processing large prey fish and relying on exclusive piscivory. the length of the intestine typically varies in the order of piscivores 12 times heavier than females). experimental and theoretical tests suggested that the large male size of this species was determined by the ability to carry empty shells and intersexual selection, and experimental tests suggested that the female size was limited by the ability to spawn eggs inside the shells. size - assortative mating has been reported in a wide range of animal taxa (e.g., planarians, snails, gammarus, insects [117119 ], fishes [120, 121 ], toads, snakes, lizards, mammals, and birds) and can cause reproductive isolation between species or morphs as byproducts of differences in body size [127129 ]. in the lake tanganyika cichlid fish, divergent natural selection on body size might have been contributed to at least a part of explosive radiation of this species flock through assortative mating, and further, other various mechanisms such as low hybrid fitness and resource partitioning. further work on intrinsic and extrinsic factors that affect the body size may give deep insights into the mechanisms underlying the adaptive radiation of these fish. huber and colleagues and pollen and colleagues demonstrated that the relative development of various brain structures relates to habitat, social behaviour, resources, and environment. thus, in the tribe ectodini for example, telencephalon, that appears to be involved in a variety of tasks, such as processing olfactory, visual and gustatory stimuli, and in learning, agonistic and courtship behaviours, was larger, and hypothalamus, that appears to have integrative functions relating to feeding, aggression, reproduction, and vision, was smaller in monogamous species compared to polygamous species. gonzalez - voyer and colleagues showed that female brain size correlated with brood care type and diet type and that male brain size correlated with diet type only, suggesting that more complex diet selection and larger burden on brood care may demand larger brain size. sylvester and colleagues showed that an alternative snp in irx1b potentially causes differences in the relative size of the telencephalon versus the thalamus between rock - dwelling and sand - dwelling lake malawi cichlids. although it is not at all obvious how an increase in the size of brain would give rise to functional differences (e.g., increased cognitive abilities), the brain morphology may have played important roles in adaptive radiation of the lake tanganyika cichlids. we reviewed a trophic morphology, body shape, body size, and nervous system, morphological traits that might be causally involved in the adaptive radiation of the lake tanganyika cichlids.. the diversity of these other traits may be a result of adaptation to various environments, genetic drift, or phenotypic plasticity during development. for example, the infraorbitals (a series of bones surrounding the lower half of the eye) were shown to vary considerably in shape among the lake tanganyika cichlids (figure 3). however, it is still unknown what exactly caused the observed inter - specific differences in this morphological structure, though it has been argued that infraorbitals have the function to regulate the movement of jaws in relation to some other bones, and that the number and size of sensory pores on these bones may be associated with the noise sensitivity. some morphological characters differ at various taxonomic levels, reflecting difference in the relative time of morphological divergence. for example, the shape of infraorbitals tends to differ between tribes, suggesting that this morphology diverged during the initial radiation. the body shape and the shape of oral jaw teeth, on the other hand, tend to differ at lower taxonomic levels, such as between genera and between species, suggesting that the divergence of these morphologies reflect later evolutionary events. studies on adaptation and natural selection typically focus on traits in adult organisms, but high mortality among juveniles indicates that in addition to predation pressure strong selection pressure, competition avoidance, and resource partitioning are presumably important already early on in life (e.g.,). thus, individuals do not only have to compete for resources against heterospecific individuals but also against conspecifics. for species that comprise a number of cooccurring size - classes (many invertebrates, fish, amphibians, and reptiles) niche separation by body size differences poses a complex problem since the smallest size - classes of one species often overlap with the largest of another. since resource utilization abilities and predation risk are generally related to body size, many species undergo sometimes dramatic ontogenetic shifts in habitat use and/or food choice. thus, among fish, ontogenetic changes in resource use are nearly universal and size - related shifts in food choice have been documented in numerous species, typically with positive correlations between food size and body size (e.g., [139145 ]). these ontogenetic shifts in resource use might vastly complicate species interactions with important consequences for community dynamics, in particular in multispecies communities. if small and large species coexist, the most critical feature of this interaction is not how adults of these two species interact, but how the larger species is able to recruit through juvenile stages that are identical to the size ranges present in the smaller species. interactions of this sort might form bottlenecks in recruitment to the species, and thus adaptive fine - tuning is particularly important in juvenile stages, especially in regions / periods where / when resources are limited. ontogenetic niche shifts aid in maximizing fitness by reducing competition with conspecifics via resource segregation, by minimizing predation risk through habitat shifts, and by maximizing growth through dietary shifts. these ontogenetic shifts in resource use might be rather abrupt and are often correlated with discrete growth periods in the life history (e.g., [149, 150 ]). for many primarily piscivorous fishes the transition to piscivory is a crucial ontogenetic niche shift. typically, highly specialized piscivores are not particularly well adapted to feeding on zooplankton and benthic invertebrates, which are their predominant prey early in life [138, 151 ], and delayed shifts to piscivory can result in slow growth and increased mortality, in particular when competing for resources with specialist planktivorous species [138, 151, 152 ]. the switch to piscivory often initiates an increase in growth rate, translating into larger body size and greater survival larger individuals are typically less vulnerable to predation and are better adapted to survive periods of starvation throughout life for specialized piscivores [151, 152 ]. several factors, for example, species - specific differences in the allometry of trophic structures, hatching time and size at hatching, have been proposed to at least partially explain these inter - specific differences in the timing of the switch to piscivory [151, 153 ]. although there is a considerable amount of studies relating dietary shifts to ontogenetic changes of overall body shape in fishes (e.g., [139141, 144, 145 ]), studies that directly relate ontogenetic changes in diet and growth patterns of the trophic apparatus are scarce (e.g.,). whereas isometric growth of the lower pharyngeal jaw was reported for the lake tanganyika cichlid lamprologus ornatipinnis, a species that predominantly feeds on invertebrates and thus does not experience a drastic shift in feeding habits throughout ontogeny, hellig and colleagues showed that an allometric change in ontogenetic lower pharyngeal jaw development of lepidiolamprologus elongatus, a top predator in the shallow rocky habitat of lake tanganyika, coincides with the dietary shift to exclusive piscivory (figure 4). this observation might indicate that distinct allometry is correlated with strong specialization, but it remains to be tested whether this is a general phenomenon in trophic specialists, what is the genetic basis of such morphological changes, and to what extent differential gene expression producing differences in morphology contributes to the astounding diversity of cichlid fishes in lake tanganyika. along with the darwin 's finches from the galpagos islands (e.g.,) and the hawaiian silverswords (e.g.,) the east african cichlid species flocks represent well - established model systems for the study of adaptive radiation. numerous morphological studies on the lake tanganyika cichlid species flock have greatly contributed to the ever - increasing knowledge on the evolutionary pathways and mechanisms generating tremendous diversity within a short period of time. rapid changes in particular morphological traits allow for the rapid adaptation of cichlid fish to novel resources. the lake tanganyika cichlids are highly diverse in their morphology, and many morphological traits appear to have been concerned with adaptive radiation. clearly, more studies are required to reveal the mechanisms of adaptive radiation of these fish, but recent methodological advances, in particular in the field of geometric morphometrics, appear promising for answering a wide variety of evolutionary questions and even allow for addressing population - level questions regarding (adaptive) shape changes (reviewed in). in the present review, we only surveyed morphological studies. however, only the integration of evidence from various fields of research will significantly advance our understanding of the evolutionary mechanisms underlying the adaptive radiation(s) of (lake tanganyika) cichlid fish. for example, disruptive sexual selection on male colouration has been shown to drive speciation in some lake malawi and victoria cichlids (e.g., [161163 ]), and olfactory cues are important for mate choice in some lake malawi cichlids. thus, female choice for male nuptial colour and olfactory signals may also have played (and still play) important roles in at least a part of the explosive radiation of the lake tanganyika cichlid species flock. indeed, more than 10% of lake tanganyika 's cichlid species (e.g., cyprichromini, benthochromini, and some species of ectodini, tropheini, and bathybatini) exhibit obvious sexual colour dimorphism. however, at present there is no clear evidence for sexual selection based on body coloration being of great importance for driving rapid diversification in lake tanganyika 's cichlid species flock. however, body coloration might serve as cue for species / mate recognition and prevent inter - specific or intermorph gene flow in case of secondary contact, be it human induced or due to lake level fluctuations, exemplified by evidence for colour assortative mating among originally allopatrically distributed colour morphs of tropheus moorii [165167 ]. thus, allopatrically evolved mate choice cues serve as prezygotic isolation mechanisms preventing cichlid species / morphs from hybridization in the case of secondary contact. recent paleolimnological and geological studies have shed light on the dynamics of past water level fluctuations that act as species pumps by recurrent fragmentation and secondary admixis of populations in the east african great lakes [169171 ]. ecological studies provide insights into the degree of intra- and inter - specific interactions (e.g., [172179 ]), and the recent developments in sequencing techniques allow for the generation of huge amounts of sequence data at comparatively low costs provide exciting new possibilities to investigate phylogenetic relationships among taxa, population genetic structure, and the genetic basis and regulation of particular traits. at present, we are still far away from completely understanding what has driven and still drives the rapid diversification in east african cichlid species flocks, but recent advances in various fields of research hold a promising future for researchers. | lake tanganyika is the oldest of the great ancient lakes in the east africa. this lake harbours about 250 species of cichlid fish, which are highly diverse in terms of morphology, behaviour, and ecology. lake tanganyika 's cichlid diversity has evolved through explosive speciation and is treated as a textbook example of adaptive radiation, the rapid differentiation of a single ancestor into an array of species that differ in traits used to exploit their environments and resources. to elucidate the processes and mechanisms underlying the rapid speciation and adaptive radiation of lake tanganyika 's cichlid species assemblage it is important to integrate evidence from several lines of research. great efforts have been, are, and certainly will be taken to solve the mystery of how so many cichlid species evolved in so little time. in the present review, we summarize morphological studies that relate to the adaptive radiation of lake tanganyika 's cichlids and highlight their importance for understanding the process of adaptive radiation. |
micrornas (mirnas) are small, noncoding oligoribonucleotides of ~21 - 22 nt which regulate gene expression through the assembly of an rna - induced silencing complex (risc). in particular, the downstream effects of mirnas relate to the fate of target mrna, which may be subjected to endonucleolytic cleavage, enrolled into a faulty translational process or, as surprisingly shown in most recent studies, translationally enhanced [112 ]. each of the hundreds of mirnas present in mammalian genomes can potentially modulate an impressively large number of target genes, thereby depicting a highly versatile network with the capacity to effectively control and modify the biochemical wiring and, in turn, the phenotypic outcome of a cell [1, 8 ]. it is now well established that mirnas are involved in disparate physiological functions, such as developmental transitions and neuronal patterning, apoptosis, fat metabolism, and regulation of hematopoietic lineage differentiation. for example, mirnas are key regulators of the nervous system in the worm and brain morphogenesis in the fish and show distinct expression patterns during mammalian brain development. a clear understanding of the functional impact of mirnas on brain neurodegeneration is an intriguing, yet rather elusive, matter of study. however, the current literature shows clear evidence that tightly controlled mirna expression is required for proper neurodevelopment and, conversely, that specific mirna dysregulation is likely linked to the pathogenesis of neurodisorders. biogenesis and silencing mechanisms of mirnas were recently revisited by carthew and sontheimer, who have highlighted common themes and unique features of both mirna- and sirna - related pathways (see figure 1 and). in either context, the molecular events that span from mirna transcription towards rna degradation are complex and imply an intricate interplay of molecular events to ensure accurate and efficient regulation of gene expression. in mammals, 80% of mirna genes are located within introns of longer primary transcripts that can be either protein - coding or mrna - like transcripts ; the majority of these are produced by rna polymerase ii [1720 ], while a minor group of genes, characterized by alu sequences, is instead transcribed by pol iii. thus, pol ii - associated transcription factors may regulate the expression of the majority of mirna genes in a tissue- and/or cell - specific fashion. while transcription of intergenic mirna genes implies usage of own promoters, intronic mirnas are transcribed with their host genes and seem to be cotranscriptionally processed prior to the removal of the host intron. typically, primary mirna transcripts or pri - mirnas are composed of a double - stranded stem of 33 base pairs, a terminal loop, and two flanking, single - stranded segments which are subject to cleavage, in the nucleus, by a protein complex called microprocessor. this is composed of a nuclear member of the rna iii family (drosha) associated with a cofactor (dgcr8) for efficient and precise processing of pri - mirnas into 6070 nt, hairpin - like precursor mirnas (pre - mirnas) [2327 ]. interestingly, several pre - mirnas, known as mirtrons, originate directly from the splicing of pri - mirnas and are subsequently processed without a requirement for microprocessor activity. evidence suggests that this alternative pathway, although rather uncommon, has emerged throughout metazoans prior to the advent of drosha [2830 ]. through the exportin-5 pathway, pre - mirnas are then transferred to the cytoplasm where they are further processed by dicer, a second rnase iii complexed with the human immunodeficiency virus transactivating response rna - binding protein, trbp [31, 32 ]. dicer binds the 3 overhang of the dsrna and then excises the terminal loop to produce a mature, single - stranded mirna duplex of approximately 22 bp. this duplex is ephemeral, in that it is rapidly unwound as soon as it associates with an argonaute protein (ago). only one strand of the original dsrna molecules is incorporated into risc while the ejected strand, unlike during the sirna unwinding mechanism, is not degraded by the associated ago [7, 1432 ]. finally, mirnas trigger gene silencing through partial base - pairing with the 3-utrs of protein - coding mrnas, thereby preventing translation of targeted mrnas and/or accelerating their degradation [15, 33 ]. the existence of stringent regulatory mechanisms affecting the biogenesis of mirnas suggests that this pathway plays a crucial role in the control of gene expression and, further downstream, the definition of biological outcomes. in this regard, several examples of double - negative feedback loops have been described, showing that the expression of mirna genes can be controlled by their own targets. in drosophila, multiple mirnas interact with different 3 utr binding sites to play a cooperative role in the posttranscriptional regulation of nerfin-1, a nuclear regulator of axon guidance, within both the developing central nervous system (cns) and peripheral nervous system. in species of this organism, the high degree of evolutionary conservation of mirna - binding sites provides evidence that regulation of the onset and extinction dynamics of nerfin-1 expression is common to all members of the drosophila genus. as shown in table 1, the effects of mirna - mediated modulation of gene expression during multiple steps of neuronal development, from early neurogenesis to synaptogenesis, have been well documented across the animal kingdom [3445, 4761 ]. strong evidence for a biological role of mirnas in neural development emerged from their identification within the hox gene clusters. when ectopically expressed, these mirnas induce a homeotic mutant phenotype, as shown in drosophila for mir - iab-4 - 5p, which reduces endogenous ubx protein levels, causing halteres to be transformed into wings. while mir-196, encoded at three paralogous locations in the a, b, and c mammalian hox clusters, directs the cleavage of hoxb8 mrna and, apparently, downregulates hoxc8, hoxd8, and hoxa7. the capacity of mirnas to directly control cell fate decisions and, in turn, specify neuron identity was also shown in caenorhabditis elegans [37, 38 ]. in developing axons, mirnas may regulate pathfinding, the process by which the circuitry of the nervous system is built. in zebrafish, normal brain morphogenesis is disrupted in the absence of the mirna - processing enzyme dicer, while the observation that functional risc complexes can be assembled in rat drg axons and growth cones is indicative of important roles played by mirnas in the regulation of axonal mrna translation. indeed, the cns displays a substantial enrichment of mirna species, of which a considerable number is expressed in a temporally- and/or spatially - controlled fashion, thereby suggesting biological implications for specific developmental stages [52, 53 ]. expression profiling revealed that several species of mirnas, such as mir-9, mir-124, mir-124a, mir-125b, mir-127, mir-128, mir-132, mir-219, and members of the let-7 family, are especially localized in the mouse brain [4045, 5461 ], while the expression of 63 additional mirnas appears to be widely distributed, although differentially, throughout the cns. of these, some are primarily present in the cerebellum (mir-195, mir-497, and mir-30b), others in the medulla oblongata (mir-34a, mir-451, mir-219, mir-338, mir-10a, and mir-10b), while a third group (mir-7, mir-7b mir-218, mir-221, mir-222, mir-26a, mir-128a / b, mir-138, and let-7c) appears to be restricted to the hypothalamus [6366 ]. in general, region - specific enrichments reflect expression rates threefold higher compared to average mirna levels displayed throughout the cns [6770 ]. conceivably, mirnas affect patterning mechanisms that specify the fate of neural cells at specific times and within proper locations. for example, an investigation of the expression of 104 mirnas during murine brain development showed that these were distributed according to specific temporal expression patterns ; in particular, the expression of 12 mirnas was significantly upregulated during embryonic stages while markedly decreased during brain development. the involvement of modulated mirnas was recapitulated by computational screens aimed at target identification, which revealed that 10 of 12 mirnas are likely associated with neurogenesis. in some instances, for example, mir-124 controls neurite outgrowth in differentiating mouse p19 cells and stimulates neuronal differentiation in the developing chick spinal cord by counteracting the antineural activity of one of its targets, namely, the small c - terminal domain phosphatase 1 (scp1). furthermore, functional studies in vivo have recently demonstrated that mir-124 controls adult neurogenesis in the mouse subventricular zone via a time - regulated control of neuroblast generation from transit - amplifying precursors. in particular, neuronal differentiation is promoted through downregulation of the transcription factor sox9, shown to be one of the targets of mir-124 [41, 72 ]. a second example relates to mir-132, which oversees dendritic morphogenesis by inhibiting translation of the synaptic protein p250gap, suggesting a key role of mir132 p250gap pathway in synapse growth and plasticty [42, 43, 73 ]. additionally, mir-133b regulates maturation and function of midbrain dopaminergic neurons through a negative feedback affecting the paired - like homeodomain transcription factor pitx3, while mir-134 activity leads to dendritic spine development through downregulation of the lim domain kinase-1. mirnas may also play significant roles in apoptosis, which is crucial in neurogenesis and during the subsequent, continued expansion of the brain size following a massive loss of neurons (i.e., 20%80%) typical of embryonic development. it was proposed that several aspects of neuronal function, for example, the control of plasticity, are directly mediated by mirnas. instead, not much evidence has yet become available to define the impact of mirnas on neural induction, namely, the stage when embryonic cells assume a neuronal identity. however, in light of a specific expression in stem cells, it is possible that mirnas play a role in self - renewal and differentiation events through the regulation of key genes, as suggested by the ability of embryonic stem cell - specific mirnas to enhance murine stem cell reprogramming [77, 78 ]. as regards human mirnas, the mirbase sequence database of the sanger center (release 14.0, dated september 2009) contains 706 sequences (http://microrna.sanger.ac.uk/sequences/). it was estimated, based on high - throughput sequencing data, that the number of mirnas expressed in the human brain may well exceed one - thousand. interestingly, many mirnas expressed in the human brain are not conserved beyond primates, suggesting a recent evolutionary origin. although functions have been assigned to only very few brain - specific mirnas, increasing evidence suggests key roles in normal development, differentiation events, and homeostasis, as well as in related pathological conditions [11, 13, 33, 36, 46, 47, 52, 100 ]. neurodegenerative diseases result from dysfunction, progressive deterioration, and extensive loss of neurons in the central and/or peripheral nervous system [10, 100 ]. in this regard, alzheimer 's disease (ad)[101104 ], parkinson 's disease (pd) [105107 ], prion diseases, amyotrophic lateral sclerosis (als) [109, 110 ], and hereditary spastic paraplegia [111, 112 ] may have a genetic or sporadic etiology. instead, huntington 's disease (hd) [113, 114 ] and metabolic disorders with neurological involvement, such as the gm2-gangliosidoses [115119 ], can only be genetically transmitted. there is now compelling evidence that dysregulation of mirna networks is implicated in the development and onset of human neurodegenerative diseases (see table 2 and [12, 120 ]). this, in turn, may provide the opportunity to elucidate underlying disease mechanisms and open up novel strategies for therapeutic applications. ad is the most common form of dementia. while several hypotheses have been proposed to explain the disease 's etiology, the causes of ad and means of stopping its progression are still elusive matters [101104, 121125 ]. features of the disease encompass neuronal loss, intraneuronal neurofibrillary tangles (i.e., aggregates of the microtubule - associated protein tau following hyperphosphorylation), and extracellular deposits of amyloid plaques (i.e., deposits of a-peptide) [102, 121125 ]. only 10%15% of ad cases represent an inheritable disease which follows an autosomal dominant mendelian pattern, while the majority arise sporadically. apparently, the disease may be caused by a genetic predisposition, as shown by the identification of specific dna mutations in a large number of families [101, 122125 ]. despite a variable etiology, a common pathogenetic cascade resulting from distinct gene defects and/or unknown environmental factors can not be ruled out. for example, accumulation of the a peptide, the cause of which is unknown, is consistently observed. in approximately 30% of sporadic ad patient samples, the expression of bace1 protein, a secretase associated with the formation of a-peptide, is significantly increased. in ad, several mirnas exhibit abnormal expression levels, suggesting a dysfunctional orchestration of gene expression [79, 80, 127, 128 ]. have recently found that mir-298 and mir-328 bind to the 3-utr of bace1 mrna, thereby producing a regulatory effect on enzyme expression in cultured neuronal (n2a) cells. presence of both mir-298 and mir-328 in the hippocampus of appswe / ps1 mice, a well - documented model for ad, and the observation that their levels of expression decrease with aging suggest that altered levels of these mirnas may deregulate bace1 and, in turn, lead to increased a formation and disease progression. moreover, bace1 can be controlled by the mir-29a / b-1 cluster, consistent with their inverse pattern of expression observed in sporadic ad patients ; in addition, a causal correlation was shown in vitro between this cluster and the appearance of the a peptide [12, 79, 80 ]. mirnas may also be involved in the neuroinflammatory process associated with deposition of the a-peptide. in this regard, the nf - kb - sensitive mirna-146a, which targets complement factor h, an important repressor of inflammatory responses in the brain, was found to be up - regulated in ad. finally, a recent work from carrettiero. shows that mir-128a regulates the cochaperone bag2 and, in turn, a pathway of degradation for microtubule - associated tau proteins with a propensity for misfolding. bag2 would normally direct tau toward an ubiquitin - independent pathway and selectively reduce the levels of sarkosyl - insoluble protein. thus, the observation that mir-128a is upregulated in ad may highlight a molecular mechanism that underlies tau inclusions in neurodegeneration. taken together, these findings suggest a mechanistic involvement of mirnas in both the amyloid and tau hypotheses for ad pathogenesis. pd is the second most common neurodegenerative disorder, characterized by resting tremor, muscular rigidity, bradykinesia, and impaired balance and coordination [105107, 130132 ]. typical pathological features are loss of dopaminergic neurons in the substantia nigra (sn) and presence of lewy bodies, which consist of intracellular inclusions affecting surviving neurons in various areas of the brain [130132 ]. these include park1 and park4 (due to a mutation or a triplication of the -synuclein gene [snca ] on 4q21 and 4p15, resp.), park3 on 2p13, park5 (due to a mutation in the uchl1 gene) on 4p14, park8 (due to a mutation in the lrrk2 gene) on 12q12, park10 on 1p, park11 on 2q, and park13 (due to a mutation in the htra2 gene) on 2p12 [105107, 133, 134 ]. the competence of embryonic stem cells to differentiate into midbrain dopamine neurons in vitro was shown to be disrupted by dicer deletion and subsequent suppression of mirna biogenesis, suggesting a physiological role for mirnas in cell differentiation and/or survival. these results were confirmed in vivo, using mice conditional for dicer, which exhibited impaired locomotor activity that recapitulated motility problems observed in pd patients. through a subtractive approach, performed by comparing mirna expression profiles in normal human adult versus pd patients midbrains, it was shown that mir-133b is specifically missing in pd and that, based on both overexpression and inhibitory tests in vitro, is likely implicated in the maturation and function of dopaminergic neurons [44, 83 ]. a markedly reduced expression of mir-133b was found in aphakia mice, a dopaminergic neuron deficiency model, which lack pitx3, a homeobox transcription factor required for neuron survival and normal motor activity susceptible to polymorphisms associated with sporadic pd. together, these observations suggest a relationship between mir-133b and pitx3, which operate through a negative feedback loop, wherein pitx3 promotes the expression of mir-133b that, in turn, downregulates pitx3. while these results point to a functional role of the mir-133b / pitx3 system in ensuring correct dopaminergic function, mir-133b knock - out mice, which are currently unavailable, would establish the extent of mir-133b impact on pd etiology. on the other hand, a more recent study showed that deletion of dicer in dopaminoceptive neurons of the murine striatum led to aberrant anatomical features (smaller brain, reduced neuron size, astrogliosis) and motor impairments (clasping and ataxia) but, surprisingly, not neurodegeneration. as dysfunction, but not necessarily loss, of dopaminoceptive neurons was previously implicated in pd, these observations, taken together, suggest that the link between dicer, mirnas, and neurodegeneration is restricted to dopaminergic neurons, thereby pointing to distinct functional roles in dopaminoceptive cells. found that in pd brains and in vitro cell models disruption of the binding site for mirna-433 led to increased translation of fibroblast growth factor-20 (fgf20). notably, an fgf20 polymorphism at 8p21.322 was previously identified as a pd risk factor correlated with increased -synuclein expression, and consequently pd onset. spinocerebellar ataxia type 1 (sca1), which is caused by the expansion of a cag repeat encoding glutamine within the gene atxn1, is characterized by the death of cerebellar purkinje cells. in eight - week old mice, depletion of dicer from murine purkinje neurons is irrelevant to cell function and survival, whereas 13-week - old animals are affected by a progressive degeneration of purkinje neurons leading to cell death. further, these older mice develop a slight tremor and mild ataxia, both of which worsen with advancing age. in 2008, lee. found that mir-19, mir-101, and mir-130 coregulate ataxin-1 protein levels and that their inhibition enhance the cytotoxic effects of polyglutamine (polyq)-expanded ataxin-1 in human cells. thus, mutations in the mirna binding sites or the mirna genes themselves might be linked to neurodegenerative phenotypes as a result of ataxin-1 accumulation. consistent with this possibility are earlier results showing that, in both drosophila and human cells, the elimination of mirnas via dicer mutation was followed by enhanced pathogenic polyq protein toxicity. hd is a fatal, hereditary neurodegenerative disorder characterized by involuntary ballistic movements, depression, and dementia [113, 114, 143 ]. hallmarks of hd are progressive chorea, rigidity, and frequent accurrence of seizures, emotional problems, loss of cognition, as well as atrophy of the caudate nucleus. the causal factor of hd is a gene mutation consisting of abnormally extended repeats of the cag sequence within the htt gene, which translates into a huntingtin protein containing an excessively increased glutamine segment [113, 114, 143 ]. disruption of mirna homeostasis, most likely in connection with an aberrant functionality of the transcriptional repressor rest, was recently shown to play a dynamic role in hd. in fact, levels of several mirnas with upstream re1 sites are decreased in hd patient cortices relative to healthy controls. interestingly, one of these, the bifunctional, brain - enriched mir-9/mir-9 targets two components of the rest complex : mir-9 targets rest and mir-9 targets corest [85, 86 ]. as a consequence of a markedly altered mirna expression, target mrnas are subject to dysregulated levels which, in turn, affect the physiological status of forebrain neurons [85, 86 ]. in 2005, rna interference was shown to produce therapeutically - relevant effects in hd mouse models [144, 145 ]. reported that rna interference through mirna technology, as compared to the shrna - based approach, is a more appropriate strategy for hd treatment. in particular, shrnas targeting mutant human hd transgenes were found to cause overt toxicity in the mouse striatum, whereas the same sequences introduced into artificial mirna expression constructs markedly alleviated the neurotoxic profile without compromising achievement of an efficient silencing effect on the murine hd gene homolog. the fragile x syndrome is one of the most common forms of inherited, x - linked dominant mental retardation affecting approximately one in every 4000 males and 8000 females [147, 148 ] with reduced penetrance of 80% and 30%, respectively [148, 149 ]. the clinical presentations of fragile x syndrome include mild to severe mental retardation, that is reflected by iq values ranging between 20 and 70, some abnormal facial features affecting jaw and ears as well as macroorchidism in postpubescent males. the gene responsible for the fragile x syndrome, fmr1, encodes a protein, fmrp, that interacts with target rnas and is implicated in mrna transport and translational control. in particular, fmrp is linked to the mirna pathway in light of its association with risc, as shown in drosophila [151, 152 ], and with argonaute proteins, dicer and mirnas, as shown in mammals [153158 ]. indeed, fmrp can act as a mirna acceptor for dicer and facilitate the assembly of mirnas [154, 159, 160 ]. thus, the neurodegenerative outcome caused by mutations in fmr1 may give rise to a host of secondary effects mediated by the action of fmrp on associated rna targets. the molecular mechanisms which underlie the pathogenesis of this disorder have yet to be elucidated. however, xu. reported that mir-124a, a nervous - system - specific mirna, is modulated, at least partially, by the drosophila homolog of mammalian fmrp (dfmr1), which was found to associate with mir-124 in vivo. that fmrp could utilize specific mirnas to regulate the translation of target mrnas was also confirmed by a recent drosophila study, in which the bantam mirna was shown to interact with dfmr1 to regulate the fate of germline stem cells. further, mir-184 was found to be repressed by mecp2, a protein that binds to methylated dna forms and plays an important role in synaptic plasticity. this observation points to a link between mirna and dna methylation pathways in the dysregulation of synaptic plasticity, a feature for which there is growing evidence of an important role played by mirnas and that is observed in the fragile x syndrome. the paradigm for a disease caused by a specific mirna is the g to a transition in the 3 utr of the myostatin / growth differentiation factor 8 gene in texel sheep. this mutation creates a target site for mir-1 and mir-206, which are highly expressed in the skeletal muscle. the downstream effect is the translational inhibition of the myostatin gene, which normally limits muscle growth but in the sheep contributes to muscular hypertrophy. based on this finding, it may be postulated that a search of human snp databases will reveal mutations that are potentially able to create or destroy putative mirna target sites and thereby contribute to phenotypic variation. one such example is a rare sequence variant of slit and trk - like 1 (slitrk1), a candidate gene for tourette syndrome located on chromosome 13q31.1 which is involved in neural development. two independent instances of the same mutation in the binding site for the mirna hsa - mir-189 were detected among a population of unrelated individuals with tourette syndrome, while absent in 3600 control chromosomes. that this mutation may be implicated in tourette 's syndrome is supported by circumstantial evidence showing an overlapping expression pattern of slitrk1 mrna and hsa - mir-189 in several brain regions implicated in the disease. several studies found that a high proportion of genomic loci containing mirna genes exhibit dna copy number alterations in common cancers and mirna misexpression has also been described in tumours of the nervous system (see table 2 and [8892, 96, 167170 ]). mirnas have been shown to act either as tumor suppressors or oncogenes and, depending on the mrna target, may accelerate the oncogenic process. a suppressor effect was observed in pituitary adenomas, the most common tumors of the central nervous system, in which down - regulation of mir-15a and mir-16 correlates with tumor size [9395 ]. other mirnas, such as the mir-155 and mir17 - 92 cluster, have an oncogenic effect [171, 172 ]. the consequence of an upregulation of mir-21 has been characterised in glioblastoma tumor cells, wherein the knockdown of mir-21 led to increased apoptotic cell death, suggesting that this mirna may act as an antiapoptotic player [88, 96, 173 ]. in addition, mirna profiling in glioblastoma cells has shown high levels of mir-221, mir-128, mir-181a, and mir-181b and low levels of mir-181c [88, 97, 98 ]. down - regulation of mir-9 and mir-125a was observed in aggressive brain malignancy, which results in the activation of medulloblastoma cell growth and arrest of apoptosis by activation of the proproliferative truncated trkc isoform. based on these findings, the potential to modulate multiple messages at the same time via mirna technology would therefore represent an intriguing prospect for cancer treatment. in addition to the canonical role as an intermediate carrier of information, this molecule may in fact perform catalytic, structural, and regulatory tasks. hence, over the last decade, unravelling the unique versatility of rna has renewed impetus towards the concept of an rna world, which refers to a self - sustaining replication system, antecedent to dna and proteins, that was engaged during a hypothetical stage at the origin of life [174177 ]. along with the most recent, stunning advances in rna biology on several fronts, the discovery of gene expression regulators has opened up a large window into the rna world. three main categories of small rnas, namely, short - interfering, micro- and piwi - interacting rnas, have emerged as regulatory players within a structurally and functionally sophisticated, and to some extent overlapping, context [14, 178 ]. unlike most of the sirnas, which silence the same locus from which they derive, the effect of mirnas is to repress genes unrelated to their own loci. thus, mirnas are subject to precise sequence requirements for the necessary interaction with heterologous targets. several approaches exist that can be employed to obtain comprehensive mirna profiling in cells or tissues ; however, the significance of a specific profile may be difficult to interpret, in light of the hundreds of target sequences in the human genome that may be associated with any particular mirnas. in this regard, computational predictions and simulations have a fundamental impact on experimental mirna research, considering that the downstream effect of a given mirna will result from the complex modulation of multiple targets along different pathways prone to cross - talk. conceivably, experimental and bioinformatic models will continue to evolve to offer large - scale screenings for the identification of the most likely mirna target(s) under a specific developmental, physiological, environmental, or pathological status. currently, functional characterization of specific mirnas is facilitated by the existence of first - class reagents such as mirna mimics and inhibitors, available through several specialized vendors. these reagents, appropriately modified to optimize correct strand utilization by risc (mimics) and ensure tight binding (inhibitors), can be used to either increase or decrease the activity of specific mirnas. corresponding applications can be exceptionally informative with respect to studies on gain (loss)-of - function effects, development of high - throughput screens to select species involved in normal and pathological cellular pathways, and the identification of targets. however, despite the significant progress in mirna research in the field of neurodevelopment and neurological diseases, it is still elusive as to whether any of the mirnas implicated in a neuropathological process is directly involved in the etiology or progression of the disorder. indeed, aberrant expression of a mirna could simply be circumstantial. this causality issue can be addressed, for example, through an accurate determination of the frequency of specific mirna mutations, the definition of temporal and spatial mirna profiles within multiple pathways in vitro, and the development of appropriate in vivo models. based on their functional role in fine - tuning metabolic pathways and genetic networks, mirnas appear to be suitable tools for use in diagnosis, prognosis, and therapy. initially, this problem was common to all rna - based therapeutics, including antisense oligos and sirnas (reviewed in). however, second - generation antisense technologies have shown that drug delivery issues can be overcome, as shown by systemic drug distribution following subcutaneous administration. specific antisense oligos called antagomirs could be used to affect the activity of mirna. in this regard, treatment of a mouse model of heart disease with an antagomir against mir-21 prevented heart failure, and antagomirs to target glioma angiogenesis has recently been proposed. mirna - based therapeutics have great potential because of their capability to efficiently silence multiple messages concurrently within an entire disease pathway. instead, conventional therapies directed at single targets require administration of a plurality of drugs giving rise to complex drug interaction and patient compliance issues. | micrornas (mirnas) have rapidly emerged as biologically important mediators of posttranscriptional and epigenetic regulation in both plants and animals. mirnas function through a variety of mechanisms including mrna degradation and translational repression ; additionally, mirnas may guide gene expression by serving as transcription factors. mirnas are highly expressed in human brain. tissue and cell type - specific enrichments of certain mirnas within the nervous system argue for a biological significance during neurodevelopmental stages. on the other hand, a large number of studies have reported links between alterations of mirna homeostasis and pathologic conditions such as cancer, heart diseases, and neurodegeneration. thus, profiles of distinct or aberrant mirna signatures have most recently surged as one of the most fascinating interests in current biology. here, the most recent insights into the involvement of mirnas in the biology of the nervous system and the occurrence of neuropathological disorders are reviewed and discussed. |
dental implants and abutments are generally produced from titanium because of its well documented biocompatibility and mechanical properties1. while the quality of implant materials made of titanium is unanimously acknowledged, the selection of suitable alloys for suprastructure is open to discussion. this selection must be done by considering the given alloy 's resistance to corrosion when coupled with titanium, its biocompatibility, and the existing clinical studies of the relation between the metals and epithelial / subepithelial connective tissues21. a galvanic current occurs when different metals come into contact with each other in an electrolytic medium, resulting in corrosion. the intensity of the galvanic current depends on different factors such as the electrode potential, polarization, surface area ratio of anode / cathode, distance between the electrodes, structure of the electrode surface, aeration, temperature, ph and composition of the electrolyte. in addition, there are some corrosion types to be taken into account, like the occurrence of crevice corrosion originating from its geometry17,21. depending on the electrode potentials of the metals in the electrolytic medium, one acts as the anode and the other as the cathode. the more noble metal is the cathode and the more active one is the anode. corrosion is expected, but in practice, the reaction does not occur or occurs at a very slow rate due to the formation of a passive layer. the corrosion rate of some metals is considerably low by reason of this passive layer formation. oral galvanism takes place when there is a difference in the electrical potential between dissimilar restorative metals5. with metals and alloys, when the materials in the oral cavity undergo corrosion, deterioration occurs in the mechanical and aesthetic properties. more importantly, corrosion products have local or systemic adverse effects on biological structures23,26. besides the extended presence of corrosion reactions can result in fractures at the interface of the alloy and abutment. some researchers have reported that high stress concentrations between the suprastructure - implant interfaces may be responsible for implant failure and the stress corrosion fractures there may evolve13,24. the effects of galvanic corrosion over short or long periods could be decreased with the use of the appropriate suprastructure alloy13. it can be said that, in the ideally selection of an suprastructure alloy, the alloy should not cause galvanic corrosion when coupled with titanium therefore no ion release occur because of the galvanic corrosion at the titanium implant or at the alloy. even though numerous studies have been conducted, all of the mechanisms involved in such corrosion are not yet completely understood12. on the one hand, in vivo evaluations are not reproducible ; on the other hand, the physiological conditions of in vitro experiments can not be fully simulated. in order to measure how corrosion - resistant dental alloys are qualitatively, electrochemical tests such as open circuit potential measurements and potentiodynamic polarization tests can be used15. electrochemical potentiodynamic polarization techniques are often used, providing general information about the corrosion resistance and susceptibility, such as the general corrosion rate, the range of passivation, and the break - down potential. in these methods, corrosion curent (icorr) and potential (ecorr) can be obtained by extrapolating the anodic and cathodic branches of potentiodynamic polarization curves. electrochemical methods have been successfully implemented in several investigations of various corrosion problems in dentistry. the aim of this in vitro study was to investigate and compare the electrochemical interaction of four different commercially available dental casting alloys when used as implant suprastructure, while coupled with titanium implant. in this study, 4 straight swissplus (sulzer dental inc., carlsbad, ca, usa) implant fixtures 10 mm in length, 4.1 mm in diameter and 4.8 mm in platform diameter were coupled with four ucla type suprastructures made of four different commercial dental alloys. the cervical regions of the implants had a 2.5 mm high machined surface and 0.6 mm high beveled shoulders. the suprastructures and suprastructure / implant couples were named as m1, m2, m3, m4 and c1, c2, c3, c4, respectively. four cylindrical ucla type suprastructures with a length of 6.5 mm and a diameter of 4.8 mm were obtained by using swiss plus plastic waxing coping (sulzer dental inc.) from four different types of dental alloy. each pattern was invested and melted in an induction casting machine (bego fornax 35 em, bremer, germany), based on recommendations of the alloy manufacturers. all casts were sandblasted to remove the oxide films and residual investments. finishing and polishing the suprastructure samples were screwed into the fixtures using a manual torque wrench with a force of 30 ncm. rh + ir+nb+mn+in+fe < % 1 m1 remanium cs, dentaurum, ispringen, germany. m3 cerapall 2, metalor mtaux prciux sa, neuchatel, switzerland m4 v - gnathosplus, metalor mtaux prciux sa, neuchatel, switzerland ti swissplus, sulzer dental inc., carlsbad. ca, usa the 1.5 mm thick copper wire was cleaned using concentrated nitric acid, and then covered with polymethyl methacrylate resin. the clamp shaped copper wire with both its tips open and covered with resin was resistant to distortion and also slightly flexible. the gap between the two ends of the resin coated copper wire was 3 mm shorter than the coupled samples (figure 1a). the coupled samples were tightened between the two ends of the copper wire. in this way, an electrical connection between the two ends of the sample was formed. finally, the uncovered copper, the top of the cylindrical suprastructure and the mtx textured surface of fixture were covered with epoxy resin (figure 1a). a jacketed beaker was used to maintain a constant temperature during the electrochemical measurements. the afnor type6 artificial saliva solution (k2hpo4:0.2, kcl : 1.2, kscn : 0.33, na2hpo4 : 0.26 nacl : 0.7, nahco3:1.5, co(nh2)2 : 1.5 g.l) buffered to 6.7 and exposed to open air was used as the electrolytic medium. a digital temperature controller system (model 9602 ; polysciences inc., warrington, pa, usa) was used to maintain the temperature of the system containing 125 ml of artificial saliva constant at 37 c. a potentiostat - galvanostat system (system model 660b ; ch instruments inc., austin, tx, usa) was connected to a computer and used for the purposes of the study. the electrochemical measurements were carried out in a single compartment three - electrode cell with the prepared alloy as the working electrode (1.33 cm), platinum foil (1.00 cm) as the counter electrode and the saturated calomel electrode (sce) as the reference electrode. the sce was drawn about 2 mm from the working electrode with a capillary tip (luggin capillary). the potential was applied between the reference and working electrode, and the resulting current between the working electrode and the counter electrode was recorded. the entire set up of the experiment is shown in figure 1b. prior to the potentiodynamic polarization test, the samples underwent 10 min of electrolysis at a constant potential of - 600 mv so that the effects of oxides and other impurities on the surface would be eliminated. potentiodynamic polarization curves were obtained between a -800 mv and 1600 mv potential range with a scan rate of 1 mv s. each experiment was performed with freshly prepared artificial saliva solution. at least three experiments were carried out in order to get similar potentiodynamic polarization curves for all of the samples. after obtaining the potentiodynamic polarization curves of all samples coupled with titanium, the concentration of the ions that leached into the artificial saliva solutions was estimated with icp - ms (thermo elemental x 7 icp - ms, winsford, england) in ppm (parts per million). a scanning electron microscope (sem) (jeol jsm 6400, jeol, tokyo, japan) was used to obtain images before and after the potentiodynamic polarization tests for the coupled samples, including the margins of the suprastructures and implants in order to determine resultant corrosion. the samples were washed several times with de - ionized water to remove any ions adsorbed to the surface and were kept in a nitrogen environment. following the sem imaging, the suprastructures were removed from the implants, and cleaned and polished once again. the open circuit potential vs. time (nearly 14 h) curves and potentiodynamic polarization curves of each sample were obtained separately for all the suprastructures and titanium under the same experimental conditions. open - circuit potentials (ocp) : figure 2 shows the ocp versus time curves of the suprastructures and titanium recorded during 5.0 x 10 seconds in afnor type solution at 37c. the ocp data obtained are summarized in table 2. the effect of the titanium and the suprastructure in the ti / suprastructure couple on the potentiodynamic polarization curves : figures 3a, b, c, d show the potentiodynamic polarization curves of titanium (ti), the suprastructures (m1, m2, m3, m4) and the ti / suprastructure couples (c1, c2, c3, c4) in afnor type solution at 37c, respectively. according to these curves it was observed that suprastructures contribute more to the potentiodynamic polarization curves of the implant / suprastructure couples (c1, c2, c3, c4) compared to the potentiodynamic polarization curves of the suprastructures (m1, m2, m3, m4) and titanium (ti) (figures 3a, b, c, d) because no considerable rise at the anodic current branch of titanium was observed between -60.0 mv and 1200 mv. the most important pitting potentials of the couples and their suprastructures are ; c1 : 626 mv, m1 : 630 mv ; c2 : 588 mv, m2 : 601 mv ; c3 : 779, m3 : 696 mv ; c4 : 521 mv, and m4 : 530. comparison of the potentiodynamic polarization curves of the ti / suprastructure samples : the potentiodynamic polarization curves of the couples (c1, c2, c3, and c4) are shown in figure 4. the ecorr and epotentials ; icorr and current values at 250 mv, 800 mv are also given in table 3 as obtained from these curves. ion concentration leached into the solution from the ti / suprastructure couples : after obtaining the potentiodynamic polarization curves of the samples coupled with titanium, the concentration of the ions that leached into the artificial saliva solutions were estimated with icp - ms and are illustrated in table 4. sem images : when sem images taken before and after potentiodynamic polarization curves were compared, extensive breakdowns was observed at surface of the suprastructures of samples c1 and c2. it was clearly observed that segregations extensively observed on the whole surfaces, however there were still little unaffected areas could be detected appearing as weakly attached to the surface (figures 5a, b). when surfaces of ti, c3 and c4 materials were investigated, almost no visible effect was revealed (figures 6a, b). in this study, ucla type abutment was used to connect a titanium implant body directly to a suprastructure. with ucla type abutment implants, removable crowns can be attached directly to the bodies of the implants by using screws9,29. a disadvantage while using this type of abutment is the possibility of corrosion due to the connection of two different metals and fitting problems resulted at casting29. the resistance a metal or an alloy exhibits against corrosion depends not only on its own properties but also on its interactions with its surroundings4. when metals and alloys are exposed to high temperature (0.0 c - 70 c), such as during eating, their ph changes (2 - 11)10. as opposed to liquids in other types of tissue, the concentration of electrolyte and oxygen in the oral cavity varies. for this reason ; dental implants, open to the oral cavity, moreover, saliva is exposed to open air, which contains gases like sox, nox, chlorides, oxygen and sulfide, resulting in atmospheric corrosion22. in biomedical applications, titanium is known to have good corrosion properties because of the stable, smooth and strongly adherent passive oxide film forming rapidly on the metal surface3 when the oxide layer brakes down mechanically. since resistance to corrosion is directly proportional to the oxide layer formation, a strong passive film formation on the metal surface enhances resistance to corrosion. it follows from this that the worst situation is the prevention of passive layer formation for a biomedical metallic material. the concentration of the metal ions released into the solution is related to the nature, composition and thickness of the metal oxides and adherence force to the alloy18. information about the general interaction at the surface of the alloy can be obtained by evaluating the changes at open circuit potential values. ocp curve values shifting towards to positive potentials show that the oxidation current of the alloy will reduce and form a passive surface layer. shifting the values of the curve towards to more negative potential indicates that the oxidation current density gradually increases, in other words ion release is taking place. the potential becomes constant against time and this behavior shows that electrochemical reactions at the alloy surface are in equilibrium. in this study, the potential of all samples shifted to more positive values and became constant after a period of time (figure 2, table 2). the open circuit potentials of m1 and m2 became constant after 280 and 145 min because of the slowly forming unstable oxide layer. on the other hand, m3 and m4 had open circuit potentials constant at 65 and 50 min, respectively. with the smallest difference between the constant and initial open circuit potential values, m4 was found to be the most suitable suprastructure alloy in the alloys tested in this study. according to the icp - ms results, the titanium concentration transferring to the solution from all of the ti / suprastructure couples was the almost the same. the investigation of the total ion concentrations leached into the artificial saliva solution shows that c3 (0.256 ppm) and c4 (0.227 ppm) dissolved less than the base alloys, c1 (1.75 ppm) and c2 (2.01 ppm) while the potentiodynamic polarization curves were being obtained (table 4). this is because c3 (79% pd) and c4 (86% au) contain large amounts of noble metals. on the other hand, the c1 and c2 base alloys were made up of ni, cr and co, cr respectively. it can also be said that because of the unstable oxide layer composition, a larger ion concentration transferred to the solution from the c1 and c2 suprastructure alloys. the corrosion current and corrosion potential were determined by extrapolation of the linear portions of the anodic and cathodic potentiodynamic polarization curves27. when a comparison was made between the potentiodynamic polarization curves of the suprastructures (m1, m2, m3, m4) and titanium implant (ti) with those of the implant / suprastructure couples (c1, c2, c3, c4), it was observed that the contribution to the couples was mostly from the suprastructure (figs. this was because the anodic current density of the titanium implant had a very small value at higher potentials and no rise was observed at the anodic branch. although titanium has a negative corrosion potential value (ecorr), its surface is covered with oxide layers (tio2 and a small amount of ti2o3 and tio), protecting it against corrosion10. these results are consistent with those of previous studies on the formation of a passive layer on the titanium surface, which increases corrosion strength20. the ecorr values of c3 and c4 (-160 v and -171 v) were found to be at more positive values than those of c1 and c2 (-517 v and -568 v). the current densities of c3 and c4 also had low values at 800 mv when compared with those of the other samples because of the noble metals present in c3 (79% pd) and c4 (86% au) (table 3). these results show that c1 and c2 are weaker materials when their corrosion performance is compared. the total ion concentration passing onto the solution from c1 and c2 showed that they had dissolved more than the other alloys, which is in agreement to the above result. the nickel and cobalt present in the structure of c1 formed a complex with the thiocyanate ion from saliva, and nickel could make complexes or compounds with urea16,28. thus, the formation of complexes and compounds could contribute to the weakness of the alloys. when the icp - ms and ecorr values of c1, c2, c3 and c4 were compared, it was expected that c3 and c4 would have lower current values at 250 mv and 800 mv (table 3). another reason for this expectation was the fact that c3 and c4 have large amounts of noble metals, palladium and gold, respectively. when dissolving total ion concentrations were compared, c3 was found as one of the alloys showing least dissolution (0.256 ppm). in addition, when the values of ecorr were compared, c3 was observed to oxidize at the most positive potential. in contrast to these results, c3 started to dissolve at 140 mv and had a current value of 250 mv, which was higher than expected. this could be due to the presence of the small amount of cu and sn, making the alloy vulnerable to corrosion even though it contains high amounts of a noble metal like palladium. the presence of the small amount of cu in the alloy prevents the material from being resistant against corrosion. bayramoglu also mentioned in their study that titanium dissolves less because of its oxide layer while sn and cu containing alloys dissolve more2. in this study, the sample c4 with au was found to have dissolved the least according to the icp - ms results, which was parallel with the results of the electrochemical test. c4 seems to have dissolved less when a comparison was made in terms of the i800mv values. however, the potassium thiocyanate present in the saliva solution in high concentrations is a disadvantage25. gold in c4 could form a complex with thiocyanate ions, posing a problem, as also reported for these types of solutions11. even beginning to dissolve at a more negative potential (epitting= 521 mv), which is a disadvantage for gold containing samples in artificial saliva solution, this sample was concluded to be superior to the others since it had dissolved less (i800mv=12.54 a cm). the low current density at 800 mv also confirmed that it was superior material comparing to the others. in conclusion, the formation of complexes with thiocyanate ion could contribute to the weakness of the c4 suprastructure alloy, but this alloy was appeared superior in terms of resistance to corrosion among the samples tested. a comparison of the sem images taken before and after the potentiodynamic polarization tests showed that the surfaces of the c1 and c2 suprastructures were extremely prone to corrosion. it was seen that the aggregations weakly attached to the surface had fallen off, and a strong presence of metal was observed (figures 5a, b). the segregations on the surface of the alloys showed that the corrosion had started from the grain boundaries. on the other hand, no significant change was observed in the sem images of ti, c3 and c4 before and after corrosion (figures 6a, b). under the circumstances of this in vitro experimental study it may be concluded that : 1., the titanium implant had virtually no effect on the pitting potential due to the passive layer formed on the surface of the implant., noble alloys were found to have dissolved less than the nickel - chromium and cobalt - chromium alloys. | objectives : as the choice of suprastructure alloy to be combined with titanium for the oral cavity is still a much debated issue, the aim of this study was to investigate the electrochemical interaction of the suprastructure / implant couples under the determined experiment conditions.material and methods : the potentiodynamic polarization curves and open - circuit potentials (ocp) of four ucla type suprastructures coupled with straight swiss plus implant fixtures were taken in afnor type artificial saliva solution at 37c. the concentration of ions leached into artificial saliva solutions was estimated with icp - ms. sem images of the margins of suprastructure / implant couples were obtained before and after the electrochemical tests.results:the ocp value of titanium became passive at the most negative potential. the lowest difference between the initial and constant ocp value was exhibited by the au based suprastructure. suprastructures made greater contributions to the potentiodynamic polarization curves of the implant / suprastructure couples. according to the icp - ms results, pd based and au based couples dissolved less than co - ni based and co - cr based couples.conclusions:within the conditions this study, it may be concluded that the titanium implant forms a stable passive oxide layer in artificial saliva exposed to open air and does not affect the corrosion properties of the suprastructures. pd based and au based couples have been found to be more corrosion - resistant than base alloy couples. |
mitral valve prolapse (mvp) is a primary connective tissue abnormality of leaflets, the chordae tendineae, and the annulus of the mitral valves. the prevalence of mvp in the general population ranges from 0.6% to10% and can change according to the diagnostic methods used, the diagnostic criteria, and the population assessed. mvp can be associated with ophthalmological diseases such as keratoconus (kc), chronic progressive external ophthalmoplegia, and retinal artery embolism [3, 4 ]. kc is a progressive, noninflammatory, idiopathic corneal ectasia characterized by changes in corneal collagen structure and organization [5, 6 ]. the prevalence of kc in the general population is 50230 per 100,000 (approximately 1/2000). it is most commonly an isolated condition, despite the multiple singular reports of coexistence with other disorders. commonly recognized associations include mvp, down syndrome, leber 's congenital amaurosis, and various connective tissue disorders. the corneal stroma is the main structure that provides corneal refraction, mechanical properties, and corneal transparency. aging, corneal pathologies, corneal surgery (e.g., lasik), and systemic diseases may affect corneal biomechanical characteristics by affecting this stromal structure rich in collagen connective tissue [1014 ]. given the cornea 's rich collagen connective tissue, corneal biomechanics may be affected by collagen connective tissue diseases. both kc and mvp are noninflammatory conditions, the etiology of which has not yet been clearly identified. in most cases of kc, furthermore, both diseases are associated with systemic collagen diseases such as pseudoxanthoma elasticum, marfan syndrome, ehlers - danlos syndrome, and osteogenesis imperfecta [1618 ]. corneal biomechanical properties can be assessed in vivo with an ocular response analyzer (ora, reichert ophthalmic instruments, buffalo, ny, usa). an ora provides measurements of goldmann - related intraocular pressure (iopg), corneal - compensated intraocular pressure (iopcc), corneal hysteresis (ch), and the corneal resistance factor (crf). crf is a parameter that shows the general resistance of the cornea, while iopcc is a parameter measured according to ch value. the assessment of corneal biomechanics can be used to prevent the misinterpretation of intraocular pressure (iop) measurements, the preoperative evaluation of patients for refractive surgery, and the separation of healthy and abnormal corneas [1214, 19, 20 ]. despite various studies of mvp prevalence in patients with kc only two studies of kc prevalence in patients with mvp this study therefore aimed to compare the biomechanical characteristics of the cornea in mvp patients, as well as to investigate the prevalence of kc in mvp. this prospective, cross - sectional, comparative study was performed at the ophthalmology and cardiology departments at yildirim beyazit university ankara ataturk training and research hospital. the study followed the tenets of the declaration of helsinki and was approved by the local ethics committee. all participants received oral and written information about the study and, prior to participating, each provided informed, written consent. participants were divided into three groups : the mvp group, the kc group, and the control group. the mvp group included 104 eyes of 52 patients, the kc group included 78 eyes of 39 patients, and the control group included 90 eyes of 45 participants. transthoracic echocardiography was performed by using a vingmed system v echocardiography system (ge vingmed, horten, norway) and a 2.53.5 mhz transducer in the cardiology department. two investigators performed independent echocardiographic evaluation, and in the case of any discrepancy, consensus was reached between them. diagnosis of primary mvp was based on the condition that one or both mitral leaflets prolapsed into the left atrium by passing the level of the mitral annulus by at least 2 mm along the parasternal long axis during the systolic phase [22, 23 ]. each participant received a complete ophthalmic evaluation that included the assessment of visual acuity, slit - lamp biomicroscopic examination, and fundoscopy. all participants also received a corneal topographical evaluation with a scheimpflug camera combined with placido corneal topography (sirius, version 1.2, cso, firenze, italy), which has been found to be repeatable and reliable. measurements of simulated keratometry 1 (sim - k1), simulated keratometry 2 (sim - k2), apical corneal thickness (act), and central corneal thickness (cct) were taken by an experienced examiner in the refractive surgery and kc unit according to sirius manufacturer 's guidelines. three measurements were made per eye, and the measurement with the best alignment and fixation was selected for data analysis. three high - quality (symmetric, well - defined inward, and outward applanation spike height) measurements were obtained for each eye. in the right eye of each participant, four parameters were obtained : ch, crf, iopg, and iopcc. all 52 patients of the mvp group had pure mvp (i.e., myxomatous degeneration) and no history of rheumatic heart disease, systemic collagen disease, or any other ocular disease. topography findings and clinical examination was used to classify the eye as normal, suspect kc, or kc. the eyes with normal corneal thickness (> 505 m), with no sign of ectasia in anterior and posterior corneal curvature were classified as normal. suspect kc is defined as abnormal localized steepening of the cornea or an asymmetric bow - tie pattern on corneal topographic examination, a normal - appearing cornea on biomicroscopy, and at least 1 of the following signs : steep keratometric curvature greater than 47.0 d, oblique cylinder greater than 1.50 d, an inferior - superior dioptric asymmetry difference in 1.4 to 1.9 d gradient, central corneal thickness less than 505 m, and an elevation of the posterior corneal surface [26, 27 ]. kc was diagnosed as having kc by the topographic pattern (asymmetric bow - tie pattern with or without skewed axes) and at least one kc sign in clinical examination (stromal thinning, conical protrusion of the cornea at the apex, fleischer ring, vogt striae, and anterior stromal scar). exclusion criteria were any history of corneal or intraocular surgery, history of glaucoma, unreliable corneal topography (e.g., corneal scar, history of previous keratitis, and corneal inflammation), or poor cooperation for reliable examination and heart disease other than mvp. contact lenses had to be removed at least 3 weeks prior to examination in the mvp and control groups. in the kc group, continuous variables were evaluated as mean sd in statistical analysis and were compared with student 's t - test. nominal data were analyzed by pearson 's chi - square test, and the correlation between parameters was interpreted with pearson 's correlation coefficient. any difference was considered to be statistically significant when the p value was less than 0.05. analysis was conducted by using spss version 17 (statistical package for social sciences inc., table 1 shows the baseline characteristics of the mvp, kc, and control groups. regarding age and gender, no significant differences occurred between the mvp and control groups (p = 0.053 and p = 0.6, resp.). in the kc group, the mean age was significantly less than that of the mvp and control groups (p 0.05). in correlation analysis, we found that in the mvp group, the ch and crf values were moderately correlated with cct (r = 0.535, p = 0.001, and r = 0.643, p = 0.001, resp.) the act was also moderately correlated with the ch and crf values (r = 0.462, p = 0.001, and r = 0.574, p = 0.001, resp.). furthermore, kc was found in six eyes of four patients (5.7%) and suspect kc in eight eyes of five patients (7.7%) in the mvp group. kc was found in one eye of one patient (1.1%) and suspect kc not found in the control group (p = 0.035). it is well known that human heart valves, like the cornea, are composed of collagen types i and v with a small proportion of type iii [29, 30 ]. frequently associated with myxomatous degeneration (i.e., the pathological weakening of the connective tissue), mvp is the most common cause of mitral regurgitation and affects 222% of the general population. it has been shown that myxomatous degeneration affecting the cornea shares some features with kc ; the damage is present particularly in the anterior part of the stroma, bowman 's membrane is disrupted, and stromal keratocytes are transformed into cells with myofibroblastic differentiation. since the alteration of collagen subtypes occurs in both kc and mvp, it is possible that a single event during embryogenesis affects both structures, for both the corneal stroma and the atrioventricular valves form during the sixth to seventh week of fetal life [9, 16 ]. dudakova and jirsova hypothesized that very similar changes in the extracellular matrix, particularly at the level of collagen metabolism, including lysyl oxidase (lox) impairment in mitral leaflets, may reflect an association between kc and mitral valve prolapse. as such, these findings may also indicate a very similar origin of kc and mvp. numerous studies have confirmed a statistically significant higher occurrence of mvp in kc patients, with a reported prevalence of 2366% compared to the 713% prevalence of mvp in the normal population [15, 34, 35 ]. though many studies have reported the prevalence of mvp in kc patients, to our knowledge only two studies have reported the prevalence of kc in mvp patients. lichter. studied 72 eyes of 36 mvp patients and 50 eyes of 25 control patients and diagnosed kc in eight eyes of 36 mvp patients (11.1%) and in one (2%) eye of a (4%) patient in the control group. to do so, they used a videokeratography system (topographic modeling system, tms-1, tomy computed anatomy, ny, usa), which is the most sensitive method used in kc diagnosis and also allows early diagnosis. by contrast, javadi. generally used conventional topography in their study but videokeratography (orbscan, bausch & lomb, rochester, ny, usa) in two patients suspected of having kc yet did not report any kc in 392 mvp patients. in our study, all participants analyzed with a scheimpflug camera combined with placido corneal topography, and similar to lichter. 's study, kc and suspect kc prevalence increased (13.4%) in mvp patients compared to those in the control group (1.1%). previous studies have found that corneal biomechanical characteristics measured by ora were affected by numerous corneal diseases, such as kc, fuchs ' endothelial dystrophy, and corneal edema, as well as some systemic factors, including diabetes and menstruation [36, 37 ]. however, this is the first study conducted to compare the biomechanical properties of the cornea in patients with mvp or kc and a control group. our results revealed that, in the mvp group, ch and crf values were significantly less than those in the control group. these results indicate that corneal viscous and elastic properties between normal and mvp patients differ. to distinguish normal from abnormal corneas before corneal surgery, it is important to assess this biomechanical difference. other studies have shown that, in keratoconic eyes, ch and crf values were less than those in normal eyes [10, 19, 36 ]. our results were similar to these ; in our study 's kc group, values of ch, crf, iopcc, and iopg were less than those in the mvp and control groups. these results indicate that keratoconic eyes are more elastic and less rigid than normal eyes, which emphasizes that the biomechanical characteristics of keratoconic eyes differ from those of normal eyes. also in this study, iopcc measurements did not significantly correlate with cct, which reveals that iopcc values provided by ora seem unaffected by corneal thickness. similarly, though iop measurements significantly correlated with crf, iopcc values were uninfluenced by crf, which suggests that iopcc measurements were also uninfluenced by corneal characteristics. it is known that keratoconic corneas are thinner, more fragile, and more prone to deformation than normal corneas. we want to underscore that the eyes of mvp patients are similar in some aspects to those of kc patients. due to structural similarity, corneal thickness in both mvp and kc patients can be less compared to that of normal patients. in our study, cct was less in 1/5 of mvp patients than in normal individuals (< 505 m). in mvp patients, corneal rigidity and resistance decreased as the cornea thinned. at the same time, ocular resistance was less in mvp patients ' eyes due to the combined effect of corneal thickness, ocular rigidity, and characteristics of corneal viscoelasticity. as such, for mvp patients particularly those diagnosed at an early age ophthalmologic examination and cct follow - up are clinically crucial. corneal biomechanical characteristics are clinically significant in selecting patients for corneal refractive surgery [19, 38 ]. recent reports have suggested that patients with early kc or suspect kc comprise 25% of patients who present for refractive surgery for myopia [39, 40 ]. while evaluating eye problems in mvp patients, especially when planning refractive surgery, corneal biomechanical characteristics and the association of kc with mvp should be considered. | objective. to investigate the biomechanical characteristics of the cornea in patients with mitral valve prolapse (mvp) and the prevalence of keratoconus (kc) in mvp. materials and methods. fifty - two patients with mvp, 39 patients with kc, and 45 control individuals were recruited in this study. all the participants underwent ophthalmologic examination, corneal analysis with the sirius system (cso), and the corneal biomechanical evaluation with reichert ocular response analyzer (ora). results. kc was found in six eyes of four patients (5.7%) and suspect kc in eight eyes of five patients (7.7%) in the mvp group. kc was found in one eye of one patient (1.1%) in the control group (p = 0.035). a significant difference occurred in the mean ch and crf between the mvp and control groups (p = 0.006 and p = 0.009, resp.). all corneal biomechanical and topographical parameters except iopcc were significantly different between the kc - mvp groups (p < 0.05). conclusions. kc prevalence is higher than control individuals in mvp patients and the biomechanical properties of the cornea are altered in patients with mvp. these findings should be considered when the mvp patients are evaluated before refractive surgery. |
idiopathic macular hole is a macular disease affecting mostly women after the 5th decade. before 1991, the disease was thought to be untreatable until kelly and wendel reported over 60% success after pars plana vitrectomy. nowadays, due to new surgical equipment and diagnostic tools, the success rate after macular hole surgery is reported to be about 9098% [2, 3 ]. although some authors reported satisfactory results without postoperative prone positioning however, in cases of long - lasting macular holes with a diameter of more than 400 m, optical coherence tomography (oct) and spectral - domain (sd)-oct data demonstrate that 1939% of so - called closed macular holes have, in reality, flat borders but bare retinal pigment epithelium (flat - open macular holes) [5, 6, 7 ]. in these eyes, visual acuity is usually limited (0.020.2). for large macular holes, different techniques have been proposed to improve surgical outcomes [8, 9, 10 ]. recently, a prospective randomized study presented by our group proved that the inverted internal limiting membrane (ilm) flap (inverted ilm flap) technique improves functional and anatomic results in patients with large macular holes as compared with pars plana vitrectomy with ilm peeling and air tamponade. functional outcomes were also better than those presented in earlier nonrandomized studies on silicone oil injection or mechanical bringing together of the borders of the macular hole. herein, we report a single case of failure of the inverted flap technique in a patient after tracheotomy. the upper diameter of the macular hole was 400 m, the lower diameter 1,617 m, and posterior hyaloid traction was visible (fig. six years earlier, the patient had had thyroid gland cancer, and during surgery the recurrent pharyngeal nerves were cut. upside down and could not maintain facedown positioning after macular hole surgery. because of the large size of the macular hole, the patient was advised to have macular hole surgery with the inverted ilm flap technique. this technique consists of core vitrectomy and trypan blue - assisted ilm peeling. during the circumferential peeling, the ilm was not removed completely from the retina but was left attached to the edges of the macular hole. a rolled segment of the peeled ilm was hanging in the vitreous cavity. next, a peripheral piece of the ilm was trimmed with a vitreous cutter or vitreous scissors and the central part of the ilm was left in place. the ilm was then massaged gently over the macular hole from all sides until the ilm became inverted, or upside down, such that the surface that normally faces the vitreous body now faced the retinal pigment epithelium. the macular hole was covered with the inverted ilm flap. at the end of surgery, the macular hole did not close either 1 week or 1 month after surgery (fig. this was the first case of failure after the use of the inverted flap technique, even though the borders of the macular hole were still elevated and the lower diameter shortened to 1,494 m. its reflectivity and thickness were lower than epiretinal membranes usually have. during the second procedure, it was observed that the ilm flap was lying on the retinal surface instead of being inverted. the rolled segment of the peeled ilm, which was lying on the retinal surface, was massaged gently over the macular hole from all sides until the ilm became inverted. however, a hyporeflective area was visible between the outer retinal layers and retinal pigment epithelium for about 10 months after surgery. during that time, sd - oct images revealed that the macula gradually became filled with tissue. during the final examination small defects of the junction between outer and inner segments of the photoreceptors and external limiting membrane remained paracentral to the fovea. on the inner retinal surface, a delicate hyperreflective structure can be seen even 18 months after surgery, without any evidence of epiretinal membrane formation. the upper diameter of the macular hole was 400 m, the lower diameter 1,617 m, and posterior hyaloid traction was visible (fig. six years earlier, the patient had had thyroid gland cancer, and during surgery the recurrent pharyngeal nerves were cut. upside down and could not maintain facedown positioning after macular hole surgery. because of the large size of the macular hole, the patient was advised to have macular hole surgery with the inverted ilm flap technique. this technique consists of core vitrectomy and trypan blue - assisted ilm peeling. during the circumferential peeling, the ilm was not removed completely from the retina but was left attached to the edges of the macular hole. a rolled segment of the peeled ilm was hanging in the vitreous cavity. next, a peripheral piece of the ilm was trimmed with a vitreous cutter or vitreous scissors and the central part of the ilm was left in place. the ilm was then massaged gently over the macular hole from all sides until the ilm became inverted, or upside down, such that the surface that normally faces the vitreous body now faced the retinal pigment epithelium. the macular hole was covered with the inverted ilm flap. at the end of surgery, the macular hole did not close either 1 week or 1 month after surgery (fig. this was the first case of failure after the use of the inverted flap technique, even though the borders of the macular hole were still elevated and the lower diameter shortened to 1,494 m. its reflectivity and thickness were lower than epiretinal membranes usually have. during the second procedure, it was observed that the ilm flap was lying on the retinal surface instead of being inverted. the rolled segment of the peeled ilm, which was lying on the retinal surface, was massaged gently over the macular hole from all sides until the ilm became inverted. however, a hyporeflective area was visible between the outer retinal layers and retinal pigment epithelium for about 10 months after surgery. during that time, sd - oct images revealed that the macula gradually became filled with tissue. during the final examination small defects of the junction between outer and inner segments of the photoreceptors and external limiting membrane remained paracentral to the fovea. on the inner retinal surface, a delicate hyperreflective structure can be seen even 18 months after surgery, without any evidence of epiretinal membrane formation. large, long - standing macular holes are less likely to be closed after pars plana vitrectomy. the inverted ilm flap technique was recently reported to be successful in nearly 100% of patients with macular holes with a diameter of more than 400 m. the single failures were observed in eyes in which it was impossible to perform the inverted ilm technique due to the fact that the inverted ilm flap became torn off the retinal surface during surgical maneuvers. this case presents a failure of this technique, even though the procedure was correctly performed and the inverted ilm flap was observed on postoperative sd - oct images and during the secondary procedure. the most probable cause of failure was the fact that the patient was unable to maintain facedown positioning due to a previous tracheotomy. the authors assumed that lying on the side might have been enough to obtain closure of the macular hole, but it was not. the ilm flap was inverted during surgery, but changed its position, probably during fluid / air exchange. postoperative sd - oct images showed the ilm flap lying on the retinal surface. also, during the secondary procedure it was noted and re - inverted. the position of the inverted ilm flap is much more controllable under silicone oil than fluid / air exchange. even though a high success rate was reported by some authors in patients who were not advised to maintain facedown positioning, it must be mentioned that those papers mostly describe results of surgery in stage i or ii macular holes. this may suggest that air / gas tamponade may be avoided in smaller macular holes, but it is crucial for the success of large, stage iv macular holes. it may also be true that air endotamponade is crucial for the success of the inverted ilm flap technique. one possible mechanism of explaining air / gas tamponade is that the bubble is a scaffold for cell migration to close the macular hole. it was documented by sd - oct that in most cases macular holes close during the first days after surgery. however, in some cases the closure process in prolonged, especially in large macular holes. some authors use silicone oil as endotamponade in large, stage iv macular holes, especially in secondary procedures. in this paper, we present for the first time the use of silicone oil tamponade as an adjunct to the inverted flap technique. it may be observed that beneath the silicone oil, the fragments of ilm cover the macular defect. to conclude, this report suggests that postoperative prone positioning is an important criterion for closure of large macular holes. silicone oil may be advised in some selected cases as an adjunct to the inverted ilm flap technique. additionally, postoperative improvement of visual acuity and retinal architecture may last many months after macular hole surgery. the authors declare no proprietary interests and no funding was received in support of this study. | aimto present macular hole surgery in a patient who had previously undergone thyroid removal surgery.material and methodsduring thyroid gland removal surgery, the recurrent laryngeal nerves were cut by the surgeon. therefore, the patient had to have a tracheotomy and because of this unusual situation, the patient could not breathe if lying upside - down. complete ophthalmic examination and spectral optical coherence tomography was performed in a 77-year - old woman before and after macular hole surgery.resultsthe patient was treated by the inverted internal limiting membrane (ilm) flap technique with air tamponade for macular hole closure. this technique was described to have very high success rates in large, stage iv macular holes. postoperatively, lying on her opposite side was advised. however, the macular hole remained open after this approach. because of this, another approach was undertaken. the ilm flap technique and silicone oil were applied, and the patient was positioned on her opposite side. silicone oil was removed after 3 months. eighteen months later, the macular hole remained closed. visual acuity improved from 10/200 to 20/50.conclusionthis case demonstrates that in an extremely select group of patients, silicone oil combined with the inverted flap technique may be considered for treatment of macular hole. |
the revision of dsm-4 to dsm-5 was felt due to several reasons including recent advances in neurosciences, clinical and public health ; identified problems with dsm-4 criteria ; and a desire to ensure better alignment with the international classification of diseases and its upcoming 11 edition- the icd-11. these changes have gained attention of academics and researchers globally and have been discussed at length. however, a critical review of these changes in the indian context has been restricted to only a few disorders and sections. substance use disorder section in dsm-5 includes changes in terminology ; sections and categories ; diagnostic criteria ; threshold for diagnosis ; severity ; and specifier. critical evaluation of the changes made to the section on disorders related to substance use in indian context has not been published so far. in dsm-4 tr disorders associated with use of psychoactive substances were grouped under the category substance related disorders. in dsm-5 these disorders have been categorized as substance related and addictive disorders in section ii, the change necessitated by inclusion of the category of behavioural addiction. dsm-5 has introduced three sections encompassing introduction, diagnostic criteria / codes and emerging measures / models in respective sections. dsm-4 categories of substance abuse and substance dependence have been clubbed together into a single disorder. this change is based on the rationale that reliability and validity of abuse has been found to be much lower than those for dependence ; some of the abuse criteria indicated clinically severe problems ; clinicians faced issue of diagnostic orphans when it was difficult to fit patient in either of two categories ; and factor analysis of dependence and abuse criteria suggested that criteria should be combined to represent a single disorder. some of the important changes in dsm-5 for disorders related to use of psychoactive substances have been summarized in table 1. important changes introduced in dsm-5 for disorders related to use of psychoactive substances one of the most significant changes in new classification system is abolition of substance dependence and abuse as separate categories. this change has addressed the debate on whether abuse and dependence are separate disorders or are on a continuum. also, previously there was much confusion among clinicians on dependence and addiction with many considering these terms as synonymous. this was especially so in case of opioid use for pain where dependence was often wrongly labeled as addiction. this undue and unjustified concern with the abuse liability on opioid analgesics lead to increased restrictions on morphine use in terminally ill patients. in spite of being the largest producer of opium producer in the word, prescription of opioid in cancer pain and other terminal illness remains abysmally low in india. while dependence is body 's adaptation to particular drug, addiction is much more complex phenomenon that has genetic, environmental and psychosocial factors. another contentious issue regarding use of terms addiction and addict has been the moralistic and judgmental views associated with use of these terms. addiction in day - to - day conversation (e. g. chocolate addiction) have also been cited to avoid use of these terms in medical lexicon. terms such as neuroadaptation and dependence have been used to demarcate behavioral dependence from a mere physical dependence. this is of relevance for india where individuals as well as family members with substance use disorders continue to experience stigma. further, the reduction in threshold for diagnosis will be useful in picking up milder cases that may benefit from intervention and also diagnostic orphans can now be diagnosed. this will also go a long way in offering appropriate early interventions for those in need. the addition of craving as a diagnostic criterion is another welcome change which will help strengthen the harmonization of dsm and icd as it is included as one of the criteria for dependence in icd-10. specifier for severity of substance use disorder also makes its reappearance after being dropped from dsm-3 r. these were removed in the fourth edition, that is dsm-4. for example, a comprehensive medication and non - pharmacological intervention based approach might be indicated in a case of severe alcohol use disorder. the milder variant of the same condition can benefit from brief intervention and motivational interviewing. locus of intervention (in - patient or out - patient) can also be guided by severity of the condition. with increasing use of dsm-5 in clinical practice this triage would help in optimum utilisation of limited mental health resources in developing country like india. the addition of behavioral addiction namely gambling disorder can be seen as another important paradigm shift. its introduction opens an avenue for inclusion of other behavioural addiction like sexual addiction, internet addiction, shopping addiction, etc. in future findings from these studies are likely to benefit our understanding and conceptualisation of substance use disorders as well. though dsm-5 has followed dimensional approach in replacing abuse and dependence criteria with a single substance use disorder entity, diagnosis is still largely dependent on a yes or no the decision to shift to a single diagnostic category of substance use disorder in lieu of separate categories for abuse and dependence is rooted in the one of basic guiding principle for dsm-5 that is to shift from stringent categorical approach to dimensional approach. however, in clinical practice, a syndromal model of diagnosis appears more promising which is bound to get diluted with these changes. the multi - axial system of dsm-4 tr, based on the bio - psycho - social approach has been discontinued. this move is likely to shift focus on biological factors as the major contributors to diagnostic categories, which could be detrimental to the bio - psycho - social approach to psychiatric disorders. in dsm-4 tr disorders associated with use of psychoactive substances were grouped under the category substance related disorders. in dsm-5 these disorders have been categorized as substance related and addictive disorders in section ii, the change necessitated by inclusion of the category of behavioural addiction. dsm-5 has introduced three sections encompassing introduction, diagnostic criteria / codes and emerging measures / models in respective sections. dsm-4 categories of substance abuse and substance dependence have been clubbed together into a single disorder. this change is based on the rationale that reliability and validity of abuse has been found to be much lower than those for dependence ; some of the abuse criteria indicated clinically severe problems ; clinicians faced issue of diagnostic orphans when it was difficult to fit patient in either of two categories ; and factor analysis of dependence and abuse criteria suggested that criteria should be combined to represent a single disorder. some of the important changes in dsm-5 for disorders related to use of psychoactive substances have been summarized in table 1. one of the most significant changes in new classification system is abolition of substance dependence and abuse as separate categories. this change has addressed the debate on whether abuse and dependence are separate disorders or are on a continuum. also, previously there was much confusion among clinicians on dependence and addiction with many considering these terms as synonymous. this was especially so in case of opioid use for pain where dependence was often wrongly labeled as addiction. this undue and unjustified concern with the abuse liability on opioid analgesics lead to increased restrictions on morphine use in terminally ill patients. in spite of being the largest producer of opium producer in the word, prescription of opioid in cancer pain and other terminal illness remains abysmally low in india. while dependence is body 's adaptation to particular drug, addiction is much more complex phenomenon that has genetic, environmental and psychosocial factors. another contentious issue regarding use of terms addiction and addict has been the moralistic and judgmental views associated with use of these terms. reasons such as trivialization of term addiction in day - to - day conversation (e. g. chocolate addiction) have also been cited to avoid use of these terms in medical lexicon. terms such as neuroadaptation and dependence have been used to demarcate behavioral dependence from a mere physical dependence. this is of relevance for india where individuals as well as family members with substance use disorders continue to experience stigma. further, the reduction in threshold for diagnosis will be useful in picking up milder cases that may benefit from intervention and also diagnostic orphans can now be diagnosed. this will also go a long way in offering appropriate early interventions for those in need. the addition of craving as a diagnostic criterion is another welcome change which will help strengthen the harmonization of dsm and icd as it is included as one of the criteria for dependence in icd-10. specifier for severity of substance use disorder also makes its reappearance after being dropped from dsm-3 r. these were removed in the fourth edition, that is dsm-4. for example, a comprehensive medication and non - pharmacological intervention based approach might be indicated in a case of severe alcohol use disorder. the milder variant of the same condition can benefit from brief intervention and motivational interviewing. locus of intervention (in - patient or out - patient) can also be guided by severity of the condition. with increasing use of dsm-5 in clinical practice this triage would help in optimum utilisation of limited mental health resources in developing country like india. the addition of behavioral addiction namely gambling disorder can be seen as another important paradigm shift. its introduction opens an avenue for inclusion of other behavioural addiction like sexual addiction, internet addiction, shopping addiction, etc. in future findings from these studies are likely to benefit our understanding and conceptualisation of substance use disorders as well. though dsm-5 has followed dimensional approach in replacing abuse and dependence criteria with a single substance use disorder entity, diagnosis is still largely dependent on a yes or no the decision to shift to a single diagnostic category of substance use disorder in lieu of separate categories for abuse and dependence is rooted in the one of basic guiding principle for dsm-5 that is to shift from stringent categorical approach to dimensional approach. however, in clinical practice, a syndromal model of diagnosis appears more promising which is bound to get diluted with these changes. the multi - axial system of dsm-4 tr, based on the bio - psycho - social approach has been discontinued. this move is likely to shift focus on biological factors as the major contributors to diagnostic categories, which could be detrimental to the bio - psycho - social approach to psychiatric disorders. overall dsm-5 seems to be promising in fulfilling its goal of dsm - icd harmonisation and movement towards an internationally compatible and practical diagnostic system for mental health disorders.. however, the real test of dsm-5 will be during clinical care and research. | in the most recent edition of diagnostic and statistical manual (dsm) that is dsm-5 many modifications have been made in substance use disorder section. these include changes in terminology ; sections and categories ; diagnostic criteria ; threshold for diagnosis ; severity ; and specifier. additionally, there have been certain additions and omissions from the earlier version. critical evaluation of the changes made to the section on disorders related to use of psychoactive substances in india context has not been published so far. the current paper presents a critique of the changes made to the substance use disorder section in dsm-5. the rationale for these changes put forth by dsm-5 work group on substance related disorders have been discussed. additionally, attempt has been made to highlight the possible future challenges consequent to the current nosological revision for substance use disorder category. overall dsm-5 seems to be promising in fulfilling its goal of dsm - icd harmonisation and movement towards an internationally compatible and practical diagnostic system for mental health disorders. it has increased the scope of addiction by inclusion of behavioural addiction. it has also tried to balance the categorical and dimensional approach to diagnosis. however, the real test of this newer edition of one of the most commonly used nosological systems will be during clinical care and research. this will help address the debatable issues regarding the changes that dsm-5 brings with it. |
we analyzed ahi signs and symptoms in 90 patients given a diagnosis of ahi during 20072014. as part of this confidential hiv testing program, routine, individual donation, hiv nucleic acid amplification testing (nat) has been provided to all rapid antibody negative participants since june 2007 (samples for nat are obtained at the time of rapid antibody testing) (7,10,11). ahi was defined as having a negative or indeterminate hiv antibody test result in the presence of detectable hiv-1 rna, corresponding to fiebig stages i ii, with a mean estimated date of infection within the previous 10 days (95% ci 714 days) (12). dates of infection were estimated for all recently infected patients using previously published criteria on the basis of serologic and virologic test results (13). at each patient s first visit after documentation of ahi diagnosis (median 4 days, interquartile range [iqr ] 36 days after ahi testing), we obtained blood samples for cd4 and viral load testing and collected detailed information regarding occurrence, duration, and start and stop dates for 11 signs and symptoms associated with ahi (5,14). in addition, patients who participated during 20072011 were asked if they had sought medical attention for any signs or symptoms. typical ahi (i.e., > 2 signs / symptoms) was defined according to criteria described by braun. (14). for statistical analysis,, chicago, il, usa) was used. for analysis on signs or symptoms compatible with ahi, signs or symptoms that started > 5 days before the estimated date of infection (i.e., before the 714 day 95% ci) were excluded. the university of california san diego s human research protections program approved the study protocol, consent process, and all study - related procedures. all 90 participants were male and self - identified as men who have sex with men (msm). median number of male partners reported for the previous 12 months was 20 (iqr 1431). a total of 72 (80%) patients had signs or symptoms associated with ahi that occurred within 2 weeks before undergoing nat ; of these 72 patients, 47 (52% of the study population) had ongoing signs or symptoms, while signs or symptoms had resolved by the time of testing for 25 (28% of the study population). twelve (13%) reported signs or symptoms starting after testing, while 6 (7%) reported the absence of signs or symptoms (table 1). a total of 66 patients (73% of the study population) reported headache, pharyngitis, or myalgia occurring during the 14 days before ahi testing. ahi, acute hiv infection ; iqr, interquartile range ; nat, nucleic acid amplification testing ; ns, not significant. most frequently observed signs or symptoms that occurred during the 14 days before nat or were ongoing at the time of nat were fatigue (53 persons, 59% of the study population), fever (51, 57%), myalgia (48, 53%), headache (41, 46%), night sweats (35, 39%), pharyngitis (32, 36%), and gastrointestinal symptoms (29, 32%). overall, 69 patients (77%) reported signs or symptoms that met criteria of compatibility with ahi (table 2). onset of signs or symptoms compatible with ahi occurred at a median of 5 days (iqr 08, range 4 to 15 days) after the estimated date of infection. neither viral load nor cd4 count correlated with duration or actual number of signs or symptoms. ahi, acute hiv infection ; gi, gastrointestinal ; iqr, interquartile range ; nat, nucleic acid amplification testing ; ns, not significant. defined as having started < 4 days before estimated date of infection or after the estimated date of infection. data on whether a patient sought medical attention because of signs or symptoms were available for 42 (47%) of 90 patients ; of these, 12 (29%) reported that they sought medical attention because of their signs or symptoms and 30 (71%) did not. significantly higher viral loads were observed for those who sought medical attention compared with those who did not (median 6.1 [iqr 5.76.7 ] log copies / ml vs. 4.7 [iqr 3.45.5 ] log copies / ml ; p<0.01). overall, 70 (78%) of the 90 patients fulfilled criteria for having typical ahi and 20 (22%) did not (of the latter, 14 had only 1 sign or symptom, and 6 were asymptomatic). patients with typical ahi had significantly higher viral loads compared with patients without (p<0.01). a total of 61 (85%) of 72 patients with signs or symptoms before nat testing fulfilled criteria for having typical ahi. in addition, 40 (85%) of 47 patients who had ongoing signs or symptoms at the time of ahi testing fulfilled criteria for having typical ahi at that time. we characterized signs or symptoms relative to the date of ahi diagnosis among persons seeking hiv testing in a program offering universal ahi screening. two findings are notable : 1) 52% of participants reported ongoing signs or symptoms at the time of ahi testing, and 2) 80% reported signs or symptoms occurring within 2 weeks before undergoing testing. these findings may have major clinical implications for community - based settings that restrict ahi testing to persons with ongoing signs or symptoms. this practice may be relatively insensitive in settings where msm undergo hiv screening frequently (11). our results show that expansion of ahi screening to include those with signs or symptoms during the 2 weeks before the test may increase the yield of ahi diagnoses by more than half. although our results may allow for estimation of sensitivity of signs and symptoms for ahi in persons seeking hiv testing, specificity of signs and symptoms remains unknown (in this study, signs and symptoms were not assessed in those who tested negative, and no control group was available). estimates on frequency of signs and symptoms in hiv - negative persons (i.e., specificity) ranged widely in previous studies. although in one study a specificity of 65% was estimated for influenza illness like symptoms (15), specificities ranging from 38% to 91% for recent symptoms were estimated in another study (5). limitations of those studies include the fact that exact time frames for occurrence of signs or symptoms (e.g., ongoing at the time of testing or occurring within the last 14 days) have not been evaluated, which makes comparison of results difficult. therefore, our results may not be applicable to populations other than msm, although previous studies have reported that clinical features of ahi may not differ by sex and age of patients (4). in summary, hiv diagnostic testing strategies that limit ahi testing to patients with ongoing signs or symptoms in contrast, hiv nat provided for msm who report signs or symptoms during the preceding 2 weeks (representing 80% of ahi diagnoses) may increase the yield of ahi diagnoses by more than half. | we analyzed signs and symptoms in 90 patients diagnosed with acute hiv infection in a community - based program that offered universal hiv-1 nucleic acid amplification testing. forty - seven (52%) patients reported ongoing signs or symptoms at the time of testing. another 25 (28%) reported signs or symptoms that had occurred during the 14 days before testing. |
breast cancer is the most common malignancy in women, constituting more than 25% of cancers in this group. the incidence of breast cancer around the world greatly varies, with highest rates in the north america, western, and northern europe and australia / new zealand (82.599.4 per 100,000 women). breast cancer metastasizes most often to axillary lymph nodes, but may involve any organ. metastasis to serosal surfaces involves primarily the pleural cavity [2, 3 ], and infrequently, the pericardial and peritoneal cavities. pleural effusions may occur at any point of time in the clinical course and may be the sole manifestation of metastatic disease. this condition is associated with a poor median survival of less than 1 year [5, 6 ]. in recent years, we have reported on the differential expression of metastasis - related molecules, including proteases, angiogenic molecules, signaling molecules, inhibitors of apoptosis, and transcription factors, in breast carcinoma effusions compared to patient - matched primary carcinomas [710 ]. the global view of cellular transcriptional activity using gene array technology is required to identify clusters of genetic markers that explain the complex biological processes involved in carcinoma progression to effusions. at present, only one study in which breast carcinoma effusions were compared with primary carcinomas is available. analyzed the gene expression signature of 19 effusions and compared them to 4 primary carcinomas, 8 cells lines and 4 specimens consisting of benign breast tissue. effusions could be differentiated into two categories, one resembling cells lines and expressing cd24, cd44 and cytokeratins 8, 18 and 19, the other expressing metastasis - associated genes, such as s100a4, upa receptor, vimentin and cxcr4. the present study compared the gene expression signatures of 10 effusions and 10 primary breast carcinomas. selected differentially expressed genes of pathways related to adhesion, interaction with the extracellular matrix (ecm) and regulation of the actin cytoskeleton were validated on mrna and protein level, and their clinical relevance was analyzed in a larger series of breast carcinoma effusions. our data demonstrate that in agreement with our previous observations, breast carcinoma cells in effusions are markedly different from their counterparts in primary carcinomas. this bears relevance to validation of novel therapeutic targets and stratification of patients with respect to treatment response and survival. ten effusions (7 pleural, 2 peritoneal, 1 pericardial) from patients with primary breast carcinoma (9 of infiltrating ductal type, 1 lobular) were submitted for routine diagnostic purposes to the department of pathology at the norwegian radium hospital during the period 19992005. submitted specimens were processed immediately upon arrival, and pellets were used for preparation of paraffin - embedded cell blocks and for freezing in equal volumes of rpmi supplemented with 20% fetal calf serum and 20% dmso. all specimens underwent morphological evaluation and were further characterized using immunohistochemistry, as previously detailed. all effusions had > 50% carcinoma cells of the total cellular content, ranging between 80100% of cells in 8/10 specimens. ten primary breast carcinomas of infiltrating duct type were retrieved from snap - frozen archival material stored at 70c at the department of pathology, norwegian radium hospital. morphological evaluation of frozen sections from the studied specimens was performed in all cases in order to ensure the presence of a predominant carcinoma cell population and absence of necrosis. eleven samples were prepared from each group (one effusion and one primary carcinoma with two samples each). (1) eleven pair - wise competitive hybridizations of cdna target sample from randomly chosen patients from the effusion group and sample from randomly chosen patients from primary solid tumor group. (2) six pair - wise hybridizations of cdna samples from randomly chosen patients from the effusion group and pooled reference sample from four primary solid tumors. biopsies from primary tumors and effusions were lysed using sv- total isolation kit (promega, madison, wis, usa) and total rna was extracted according to the manufacturer 's guidelines. the quantity and quality of the total rna preparations were assessed using a nanodrop nd-1000 (nanodrop, wilmington, de, usa) combined with agarose gel electrophoresis and only samples with ribosomal 28s/18s ratio near 2 were selected for array analysis. twenty micrograms total rna was mixed with 2 g oligo dt (amersham biosciences, piscataway, nj, usa) and rnase - free water to a total volume of 16.9 l, incubated 10 minutes at 75c and cooled on ice. six microliters of first strand buffer, 3 l of 0.1 m dtt, 2 l of superscript ii rt (invitrogen, carlsbad, calif, usa), 1.2 l 25x aminoallyl (aa - autp)/d - ntp mix (sigma, haverhill, uk) and 1 l of rnase inhibitor (amersham biosciences) were added to each reaction tube. additional 1 l of superscript ii rt was added following incubation for 60 minutes at 42c. free rna was disassembled with mixture of 10 l 1 m naoh and 10 l 0.5 m edta and underwent neutralization with 25 l 1 m hepes. unincorporated aa - autp and free amines were removed using microcon ym-30 spin column (millipore, bedford, mass, usa) and cdna yield were measured by nanodrop spectrophotometer. the cdna target samples were labeled by cy3 or cy5 fluorescent dyes (amersham biosciences) according to the sample group and resuspended in 0.1 m carbonate buffer (ph 8.6). the samples were incubated in the dark for 1 hour in rt and diluted in 35 l naoac 100 mm (ph 5.2). the free dyes were removed using qiaquik pcr clean up kit (qiagen, valencia, ca, usa), washed with 5 mm phosphate buffer (ph 8.0, 80% ethanol) and eluted with 4 mm phosphate elution buffer (ph 8.5). the hybridization efficiency and the yield of the dye incorporation were calculated using labeled cdna calculator (http://www.pangloss.com/seidel/protocols/percent_inc.html). equal amount of cy3/cy5 labeled cdna were mixed and dried in the speed - vac. the slides, with printed array - ready oligo set for the human genome version 3.0 (qiagen) containing 34,580 longmer probes, representing 24,650 genes and 37,123 gene transcripts, were blocked with 1%bsa in 0.1% sds 5xssc buffer, washed in distilled water and dried at 1000 rpm for 2 minutes. the mixed pair of samples was resuspended in hybridization buffer (25% formamide, 0.1% sds in 5xssc) supplied with 0.02 g t - rna and denatured at 95c. the arrays were hybridized in a water bath, in sealed, watertight hybridization chambers (dietech, ford city, pa, usa) for 1618 hours at 42c. after hybridization, the slides were rinsed in a coupling jar containing 2 ssc 0.1% sds, followed by washing for 5 minutes in 1 ssc, then for 5 minutes in 0.2 ssc, and finally for 10 minutes in 0.05 ssc. griding and analysis of images was performed using gene pix 6.0 software package (molecular devices, sunnyvale, calif, usa). the fluorescent intensities of cy5 and cy3 for each target spot were adjusted so that the mean cy3/cy5 ratio of housekeeping genes was equal to one, a design allowing more precise analysis of differentially expressed genes. statistical analysis of microarrays was preformed by the genomic data analysis unit of hadassah medical school, hebrew university of jerusalem. the quality assurance, calibration, data normalization (lowess) and volcano plot for gpr files were performed by custom built package written in matlab r2007a. an additional statistical analysis and clustering were carried out using the spotfire (somerville, ma) and partek (st. louis, mo) software packages. gene annotations and specific pathways were fingered out using online free access programs such as : go annotation (http://www.geneontology.org/), onto - express, pathway - express (intelligent systems and bioinformatics laboratory, computer science department, wayne state university). total rna from specimens analyzed for bcar1, vil2 and dcn expression was extracted using tri - reagent (sigma) according to the manufacturer 's guidelines. 0.5 g total rna was reverse - transcribed using the m - mlv reverse transcriptase (promega) with incubation of 2 hours at 37c, followed by 5 minutes at 95c, and diluted to 1 : 5 with rnase - free water. rt - pcr was performed on complementary dna samples using a dna thermal cycler (eppendorf mastercycler gradient, eppendorf, hamburg, germany) with reddymix pcr master mix (abgene, surrey, uk). sense 5-ggg - cca - cag - gac - atc - tat - gat-3, antisense 5-gag - gaa - cgt - cgt - aga - ctg - cg-3 (amplicon size, 318 base pairs [bp ]). sense 5-gtt - ttc - ccc - agt - tgt - aat - agt - gcc-3, antisense 5-tgc - ctt - tgc - aaa - gct - ttt - att - tca-3 (amplicon size, 995 bp). conditions were as follows : bcar1 : 95c for 3 minutes, denaturation at 95 for 15 seconds, annealing at 59 for 30 seconds, extension at 72 for 20 seconds, 34 cycles ; vil2 : 95 for 3 minutes, denaturation at 95 for 15 seconds, annealing at 64 for 30 seconds, extension at 72 for 20 seconds, 33 cycles. products were separated on 1.5% agarose gels, isolated using the invisorb spin dna extraction kit (invitek gmbh, berlin, germany) and sequenced. gels were photographed by the kodak edas 290 system (kodak, rochester, ny, usa). densitometer analysis of films was performed using a computerized image analysis (nih image 1.63) program. bcar1 and vil2 mrna levels were established by calculating the target molecule/28s ratio (all cases scored for band intensity compared to control). qrt - pcr was preformed using the mx3000p qpcr system (stratagene, calif, usa). oligonucleotide primers were designed in the primer express program (applied biosystems, foster city, calif, usa). primer sequences for dcn were 5-tcc - gct - gaa - gag - ctc - agg - aat-3 for the forward primer, and 5-cct - tga - gga - atg - ctg - gtg - ata - ttg-3 for the reverse primer. the primers for rplpo normalizer gene were : 5-cca - act - act - tcc - tta - aga - tca - tcc - aac - ta-3 for the forward primer and 5-aca - tgc - gga - tct - gct - gca-3 for the reverse primer. one of the primers in each primer pair was designed in exon - exon boundaries region in order to minimize the dna contamination noise. the specificity of primer binding was analyzed by blast (http://blast.ncbi.nlm.nih.gov/) with human genomic + transcript (human g + t) database for highly similar sequences (megablast). the primer optimal concentration and the sensitivity, efficiency, and accuracy of qpcr were calibrated by amplifying serial geometric dilutions of pooled sample consisted from five primary tumor and five effusion cdna samples. 0.1 g of cdna product from the reverse transcriptase reaction were amplified using dynamo sybr green qpcr kit with rox passive reference dye (finnzymes oy, espoo, finland) according to the manufacturer 's instructions. absence of primer - dimers and non - specific products was verified by single product peak in the qpcr dissociation curve. in addition, the pcr product was separated by gel electrophoresis and sequenced (hebrew university facilities). formalin - fixed paraffin - embedded sections were available from 52 breast carcinoma effusions (47 pleural, 4 peritoneal, 1 pericardial) from 51 female patients (one patient with 2 effusions) aged 3386 (mean = 59) years with histologically verified breast cancer. in 27 cases, the primary carcinoma was additionally available for analysis. slides from the primary breast carcinoma specimens were available in our archives for 45 cases. these were diagnosed as infiltrating duct carcinoma (38), lobular carcinoma (5) or mixed duct and lobular carcinoma (2). in the remaining 6 cases, these 79 above - described specimens were manually immunostained for p130cas, phospho - ezrin (p - ezrin), and claudin-4. the monoclonal mouse p130cas antibody (clone cas-14) was purchased from neomarkers (labvision corporation, fremont, calif, usa). a monoclonal mouse p - ezrin antibody was purchased from bd pharmingen (san jose, calif, usa). the rabbit polyclonal claudin-4 antibody was purchased from zymed (san francisco, calif, usa). all slides underwent pretreatment in a microwave oven for 20 minutes (p - ezrin and claudin-4 slides in tris / edta buffer, ph = 9 - 9.1, p130 slides in citrate buffer, ph = 6). visualization was achieved using the envision + peroxidase system (dako a / s, glostrup, denmark). negative controls consisted of sections that underwent similar staining procedures with isotype - matched mouse antibody, normal goat igg or non - relevant rabbit immunoglobulins according to the antibody host species. positive controls consisted of a breast carcinoma biopsy that demonstrated immunoreactivity for the studied antigens in a pilot study. staining was considered positive only when localized to the cell membrane in a linear pattern for p - ezrin and claudin-4, and when present in the cytoplasm for the p130cas reaction. staining extent was scored on a scale of 04, as follows : 0 = no staining, 1 = staining of 15%, 2 = staining of 625%, 3 = staining of 2675%, 4 = staining of 76100% of cells. slides were scored by a surgical pathologist experienced in effusion cytology and breast pathology (bd). frozen specimens were thawed and subsequently lysed in 1% np-40, 20 mm tris hcl (ph 7.5), 137 mm nacl, 0.5 mm edta, 10% glycerol, 1 mm phenyl - methylsulfonyl fluoride, 1 g / ml aprotinin, 2 g / ml leupeptin, 1 mm sodium orthovanadate, and 0.1% sds. 25 g of a total protein from each sample were separated by electrophoresis through sds-10% polyacrylamide gels under reducing conditions. after electrophoresis, proteins were transferred to immobilon transfer membranes (millipore, bedford, mass, usa). membranes were blocked in 5% non fat dry milk (nfdm) in 0.1% tween tbs (tbst) and incubated overnight at 4c in 5% bsa tbst containing anti - p - ezrin (thr) rabbit mab (cell signaling technology inc., danvers, mass, usa). after incubation, membranes were washed and incubated for 1 hour with peroxidase - conjugated affinipure goat anti - rabbit igg (jackson immunoresearch, west grove, pa, usa) in tbst containing 5% bsa. membranes were developed using the enhanced chemiluminescence kit (pierce, rockford, ill, usa), according to manufacturer 's specifications. membranes were then washed, stripped in 0.2 m glycine, 0.1% sds, and 1% tween 20 (ph 2.2), blocked in tbst containing 5% nfdm, and incubated overnight at 4c in 5% bsa in tbst containing a rabbit polyclonal anti - ezrin ab (abcam, cambridge, uk). total ezrin activity was normalized to -actin activity measured using rabbit anti--actin polyclonal antibody (cell signaling technology inc.). levels of phosphrylation of the torc1 substrate p70s6k were analyzed using goat anti - p - p70s6k and rabbit anti - p70 s6k antibodies (santa cruz biotechnology, inc. secondary peroxidase - conjugated donkey anti - goat igg (santa cruz biotechnology, inc.) was used for anti - p - p70s6k detection. densitometer analysis of films was performed using a computerized image analysis program (nih image 1.63). ihc and ib results were analyzed using the spss - pc package, version 15.0 (chicago, ill, usa). comparative analyses of tumor cell expression results in all effusions versus primary tumors were performed using the mann - whitney u test. the same test was applied for analysis of the relationship between protein expression in effusions and clinicopathologic parameters. the wilcoxon signed ranks test was applied for patient - matched analysis in the 27 cases with effusion and primary tumor. univariate analysis for disease - free survival (dfs) and overall survival (os) for 44 patients with clinical data were executed using the kaplan - meier method and log - rank test. for this analysis, expression categories were grouped as focal (25% of cells) or diffuse (> 25% of cells). we have previously shown that effusions constitute a unique form of breast carcinoma metastasis with mrna and protein expression patterns that differ from primary tumors and solid metastases [710 ]. in the present study, we compared the global expression profile of breast carcinoma cells in effusions with that of primary carcinomas. figure 1(a) shows volcano plot of global gene expression in effusions and primary tumors. differences of 15 fold in gene expression with cut - off p - value.05). os for the 44 patients with survival data ranged from 2393 months (mean = 90 months), while dfs ranged from 0336 months (mean = 55 months). in survival analysis, higher p130cas expression in effusions was associated with a trend for poor os (p =.062) and dfs (p =.098 ; figure 5). semiquantitative rt - pcr analysis of 43 primary tumors and 25 effusions showed significantly higher vil2 expression in effusions (p =.0021, figure 3(c)). protein levels and phosphorylation extent of ezrin were analyzed by western blotting using phospho- and pan - specific antibodies. pan - ezrin protein level was significantly higher in effusions (p =.004, figure 3(d)), whereas p - ezrin levels did not significantly differ. although the fraction of phosphorylated protein did not significantly differ, the total amount of the protein was up - regulated in effusions (p =.004). thus, the absolute phosphorylated ezrin levels were higher in effusions compared to primary tumors. immunostaining of 51 of the 52 effusions (one unsatisfactory reaction) and 27 primary carcinomas showed significantly higher p - ezrin expression in effusions compared to primary carcinomas (p.05). tsc1 showed a 2.3-fold downregulation in effusions compared to primary tumors in the array analysis. analysis of the phosphorylation level of the rapamycin sensitive mtor complex 1 (mtorc1) substrate p70s6k showed that in spite of the tsc1 downregulation, the levels of p70s6k phosphorylation were lower in effusions compared to primary tumors (p =.003, figure 6). breast carcinoma metastasis to the serosal cavities represents an advanced stage in tumor progression and is associated with extensive alterations at the molecular level, involving clinically established targets such as her-2 and hormone receptors, as well as other cancer - associated molecules [710 ]. despite the fact that breast carcinoma is one of the most extensively studied cancer forms, little effort has been directed towards understanding the biology of tumor cells in malignant effusions. the major aim of the present study was to characterize a general expression fingerprint that distinguishes effusions from primary tumors. our data show that 351 genes among 24,650 gene transcripts are significantly altered in effusions in comparison to primary carcinomas. many of these genes are involved in ecm - receptor interaction, focal adhesion and regulation of the actin cytoskeleton pathways, which define the metastatic potential of carcinoma cells by enhancing their motility and leading to anoikis escape in the absence of ecm molecules. the gene expression profile correlated with phenotypic change during the transition of breast carcinoma cells from the solid tumor to suspended cell clusters in pleural effusions. carcinoma cells in effusions showed down - regulated ecm encoding molecules such as decorin, fibronectin, collagens i, xxii and v, concomitantly with the downregulation of the ecm - binding receptors, integrins 5 and 7. thus, it appears as if the cells in effusions lose the requirement for interaction with matrix components, possibly by a compensatory signaling mechanism within the cells. the second goal of the study was to highlight molecules with multiple functional influences on the metastatic potential of cells in effusions. this protein provides a functional link between the plasma membrane and the actin cytoskeleton by interacting with the cytoplasmic domains of adhesion membrane proteins and regulation of cytoskeleton polymerization through the rho pathway activation. moreover, ezrin promotes growth and survival via akt / mtor pathway activation in ewing 's sarcoma cell lines. a recent study provided additional information regarding the role of ezrin in elevation of the metastatic potential of carcinoma cells, by showing that its downregulation of the cell - cell adhesion molecule e - cadherin, and suggesting that ezrin is associated indirectly with the e - cadherin/-catenin complex by regulating src activation. screening of a broad spectrum of human cancers, including breast, lung and prostate tumors showed high expression of ezrin in tumors of mesenchymal origin and in primary breast carcinomas. moreover, ezrin expression was shown to be strongly associated with poor prognosis in breast carcinoma. in agreement with the latter report, higher vil2 mrna expression in effusions was associated with poor disease - free survival in our cohort (data not shown). this finding requires further investigation, as it was obtained in analysis of only 17 effusions. the up - regulation of ezrin in effusions was validated using rt - pcr, western blotting and ihc. we found that the up - regulation of ezrin in effusions is associated with expression of the functionally active t567 phosphorylated ezrin [17, 18 ] at the plasma membrane. this gene is not only up - regulated at the mrna and protein levels, but is also more active in effusions compared to solid tumors. since the increase in ezrin activation may influence multiple metastasis - associated cell functions [17, 18 ], the therapeutic targeting of this protein may prove beneficial in effusion therapy. statistical analysis of the array results showed that the tsc1 gene, encoding a protein involved in mtorc1 inhibition, is down - regulated in effusions in comparison to primary tumors. moreover, there is evidence that mtor activation can lead to anchorage - independent growth of carcinoma cells, making it a potentially important factor for cell survival in effusions. rapamycin analogues, such as temsirolimus (cci-779) or everolimus (rad-001) that target mtor are now in different stages of clinical trials for anti - cancer therapy as a single agent or as additive treatment having synergetic effect with er- and her2/neu- targeted therapy [24, 25 ]. downregulation of the mtor inhibitor tsc1 in effusion may lead to subsequent activation of mtorc1 at this site of metastasis, suggesting that this signaling pathway may be altered along tumor progression in breast carcinoma. recent studies demonstrate that tsc1 directly interacts with ezrin and through this interaction regulates focal adhesion complex formation and causes cytoskeletal remodeling [26, 27 ]. the loss of tcs1 results in loss of focal adhesions, cell rounding and progressive detachment of cells from the substrate. since effusions are characterized as clusters of detached carcinoma cells, the parallel dysregulation of tsc1 and ezrin expression may play a critical role in effusion formation. the relative phosphorylation extent of the mtorc1 substrate p70s6k [28, 29 ] was measured in order to analyze the effect of tsc1 downregulation on the mtorc1 activity. in spite of the downregulation of the mtorc1 inhibitor tsc1, the extent of p70s6k phosphorylation remains low in the effusions in comparison to primary tumors. one possible explanation is that other substrates of mtor may be relevant and p70s6k may be a minor effector of this pathway in effusions. hormone receptor status and the relevance of adjuvant hormonal therapy at different stages of the disease are central in breast carcinoma research. we have previously shown that er is down - regulated in effusions in comparison to primary tumors. in the present study we observed bcar1 gene up - regulation in effusions. high bcar1 expression was reported to be associated with a poor response to first - line tamoxifen therapy in patients with recurrent disease and with an increased rate of relapse. moreover the up - regulation of p130cas in effusions can be a result of population enrichment by resistant cells due to tamoxifen treatment. thus, bcar1 expression status in effusions must be taken under consideration while choosing therapeutic regimen in patients with breast carcinoma effusions. the up - regulation of p130cas can lead to subsequent activation of rac pathway and actin cytoskeleton rearrangements [34, 35 ]. this can elevate the metastatic potential of the cells in effusions by enhancing cell migration and leading to anoikis escape, as has been shown in in vitro systems [36, 37 ]. in contrast to the established importance of er- as a breast cancer marker, the prognostic and predictive relevance of er- remains unclear. several previous reports have shown correlation between low er- expression and advanced disease stage and shorter survival in various tumors, including gynecological carcinomas [3840 ]. in the present study we found downregulation of er- in effusions. since breast carcinoma effusions constitute stage iv disease, this observation is concordant with the above - detailed publications. low er- levels in effusions may contribute to tamoxifen resistance, as had been shown in er - positive primary breast carcinomas. thus, the expression levels of er- may influence decisions regarding therapeutic regimens for patients with this form of metastatic disease. claudins are a family of tight junction (tj)-specific integral membrane proteins, including more than 20 members to date. tjs, located between epithelial or endothelial cells, at the apical region of the adjacent lateral membranes, control the paracellular transport of solutes and maintain cell polarity by blocking the free diffusion of proteins and lipids between the apical and basolateral domains of the plasma membrane [4244 ]. tj filaments also contain occludin, the first tj - specific integral membrane protein identified, yet it has been shown that claudins are essential and sufficient to form tj strands. the structure of claudins consists of intracellular amino and carboxy termini, four transmembrane domains, and two extracellular loops mediating interactions between claudins on adjacent cells [4244 ]. the second extracellular loop serves as a binding site for clostridium perfringens enterotoxin in claudin-3 and -4. the carboxy terminus of most claudins contains potential serine and/or thereonine phosphorylation sites and a pdz - binding motif, to which the tj cytoplasmic scaffolding proteins zo-1, -2 and -3 bind. we have recently shown that several claudin family members are upregulated in ovarian carcinoma effusions compared to corresponding primary carcinomas. in the present study, we found upregulation of claudin-4 in breast carcinoma effusions compared to primary carcinomas, suggesting that members of this family are upregulated at this anatomic site in multiple epithelial malignancies. our observations are in agreement with a recent study in which claudin-4 expression was shown to be associated with high grade and poor prognosis in breast carcinoma. the previously discovered role of claudin-3 and claudin-4 in cell motility and increased mmp-2 activity suggests that this may be yet another metastasis - promoting molecule in breast carcinoma effusions. in conclusion, gene array analysis of breast carcinoma effusions and primary carcinomas showed differences in expression of multiple genes regulating cell motility, invasion and metastasis. the study of effusions and the way they differ from solid tumors will expand our knowledge regarding tumor progression in general, as well as regarding malignancies affecting this anatomic site in particular, and may have an impact on treatment modalities and prognostic models. | the detection of breast carcinoma cells in effusions is associated with rapidly fatal outcome, but these cells are poorly characterized at the molecular level. this study compared the gene array signatures of breast carcinoma cells in primary carcinomas and effusions. the genetic signature of 10 primary tumors and 10 effusions was analyzed using the array - ready oligo set for the human genome platform. results for selected genes were validated using pcr, western blotting, and immunohistochemistry. array analysis identified 255 significantly downregulated and 96 upregulated genes in the effusion samples. the majority of differentially expressed genes were part of pathways involved in focal adhesion, extracellular matrix - cell interaction, and the regulation of the actin cytoskeleton. genes that were upregulated in effusions included krt8, bcar1, cldn4, vil2, while dcn, cldn19, itga7, and itga5 were downregulated at this anatomic site. pcr, western blotting, and immunohistochemistry confirmed the array findings for bcar1, cldn4, vil2, and dcn. our data show that breast carcinoma cells in primary carcinomas and effusions have different gene expression signatures, and differentially express a large number of molecules related to adhesion, motility, and metastasis. these differences may have a critical role in designing therapy and in prognostication for patients with metastatic disease localized to the serosal cavities. |
rhabdomyomas are the most common primary cardiac tumors in childhood.13 this neoplasm may be associated with tuberous sclerosis tuberose sclerosis which is an autosomal - dominant disaese affecting the brain, skin, kidney, heart and characterised by infantile epilepsy, mental retardation, facial adenoma sebaceum. although the indication of surgical resection of symptomatic tumors is well established,4 medical follow - up is prefered unless critical obstruction or dysrhythmias are present.5 we describe a newborn with a huge cardiac rhabdomyoma in the left ventricular outflow tract that initially produced moderate obstruction, but resolved spontaneously. a male neonate aged four hours was admitted to cerrahpa?a medical faculty because of a heart murmur detected two hours earlier. physical examination was normal except for a grade 3/6 systolic ejection murmur which was heard maximally at the left sternal border in the third intercostal space. heart rate was regular at 126/min and blood pressure was 65 mmhg in the right arm. echocardiography showed normal chamber dimensions and anatomy, along with a spherical mass originating from the mitral valve, just below the aortic valve, interposed between the ventricular septum and mitral valve anterior leaflet. multiple additional masses were present within the left and right ventricular cavities (figures 1 and 2). the location, number and size of each tumor were : mass in right ventricular apex mass below septal leaflet of tricuspid valve mass on interventricular septum doppler showed turbulence within the aorta with a systolic gradient of 30 mmhg between left ventricle and aorta (figure 4). although there were no other signs of tuberose sclerosis, the baby was thought to have tuberose sclerosis presenting with heart disease, a common presentation.6 due to the asymptomatic nature of the condition, a conservative approach was taken. serial echocardiographic studies were undertaken and at one month of age, the subaortic tumor decreased to 7 by 6.8 mm and the outflow gradient fell to 22mmhg. left ventricular outflow tract obstruction caused by mass neurologic and radiologic features of tuberose sclerosis appeared, with convulsions and subependymal hamartomas at 6 month of age. but cardiologically the patient has remained asymptomatic. follow - up investigations showed spontaneous regression of the tumors within eight months and the outflow gradient disappeared completely (figure 5). a clinicopathologic study showed that such tumors are usually multiple (92%), often intracavitary (50%), and occur more frequently in the left ventricle than in the right ventricle (100 % vs 81 %).2 cardiac rhabdomyomas are frequently associated with tuberose slerosis,6 with a prevalence of 30- 80%.13712 the other primary heart tumours at this age (hamartomas, myxomas and fibromas) differ both clinically and ultrasonographically from rhabdomyomas. as most rhabdomyomas are multiple, the diagnosis is generally beyond doubt, and may be made even in the absence of histologic confirmation.13 multiple intracavitary tumours are considered as an important marker of tuberose sclerosis, even in antenatal period.14 multiple tumors, or a single tumor plus involvement of other organs (e.g. central nervous system, kidney, skin) that are compatible with the diagnosis of tuberose sclerosis, or a single tumor with positive family history of tuberose sclerosis, is highly suggestive of rhabdomyomas.1 cardiac rhabdomyomas may be asymptomatic, or may cause a variety of clinical symptoms depending on their size and location.315 in the majority of cases, symptoms occure at an early age or even before birth.614 the spectrum of clinical manifestation ranges from cardiac murmur to sudden death.5 the presenting symptom may be arrhythmia, cardiac murmur, complete or variable atrioventricular block, pericardial effusion, cardiomegaly, cardiac failure or sudden death.361017 the variety of symptoms can be explained on the basis of obstruction of blood flow, myocardial involvement and disturbance of the cardiac rhythm. the value of surgical resection of symptomatic tumours is well established.4 while most rhabdomyomas appear to regress spontaneously, some infants may benefit from surgery for obstructive lesions at an early stage.13 with surgery, it is possible to remove obstruction or an arrhythmogenic substrate. rarely, even heart transplantation may be indicated in patient with severe myocardial involvement.18 but surgical intervention is neither possible nor indicated in every child.19 consequently, a conservative approach is preferrable and useful in most cases. unless critical obstruction or dysrhythmias is present, medical follow - up should be preferred5 since these tumours demonstrate benign pathological characteristics and tend to regress over time.11213151920 the chance of spontaneous regression does not depend on the initial size, number or location of tumours.15 partial or complete spontaneous regression of rhabdomyomas has been reported in 54% of cases.1 this regression may take place in a period as short as three weeks.1 echocardiography is useful in determining tumour size, number and location. serial echocardiographic studies are both useful and safe in monitoring tumour size, and they provide acceptable follow - up information.21 | rhabdomyomas are the most common primary cardiac tumors in childhood, and are often associated with tuberous sclerosis. we report a huge rhabdomyoma in an asymptomatic four hour old infant who presented initially with a murmur due to moderate subaortic stenosis. followup showed regression of the tumour. although the indications for surgical resection of symptomatic tumors are well established, medical follow - up should be the prefered treatment. |
rabbit anti - phospho - akt (ser473), rabbit anti - phospho - mek1/2 (ser217/221), and mouse anti - cleaved parp (asp214) were all from cell signaling technology ; mouse anti - tubulin (sigma) ; mouse anti - tom20 (bd biosciences) ; rabbit anti - gfp ; rabbit anti - eea1. wild type and activated (g12v) gfp - n - ras, gfp - er / golgi - ras (gfp - n - hvr - ala) and gfp - n - ras acceptor constructs lacking the c - terminal hvr have been previously described (laude and prior, 2008). the gfp - n - ras acceptor constructs allow subcloning of membrane targeting motifs c - terminal to the ras g domain separated by a three amino acid ara - linker sequence. 274358), the gm130 golgi targeting domain (aa 784986) and the omp25 mitochondrial targeting domain (aa 170206) from plasmids encoding the relevant proteins : fens_fyve_f : 5-actcgaggtgagctcagcagagaagaggctcct-3 and fens_fyve_r : 5-ctcgaggcccccaccgccacgagaagtccgatcttcatctttgatggagtc-3. gm130_f : 5-ctcgagctgctcacttggcagcccca-3 and gm130_r : 5-gaattcttatataaccatgattttcacctcgtcgttctc-3 ; omp25_f : 5-ctcgaggacatcgaggcgacggagaggcc-3 and omp25_r : 5-gaattccctcagagctgctttcggtatctcacgaaggc-3. the sequenced targeting motifs were subcloned into the n - ras acceptor construct and correct targeting confirmed in hela cells by immunofluorescence microscopy. hela cells were grown at 37 c in dmem (invitrogen) supplemented with 10% heat - inactivated fbs. nih3t3 flp - in cells (invitrogen) were grown in the presence of 100 g / ml zeocin. for the stable expression of compartmentalized ras, nih3t3 flp - in cells were transfected with pef5/frt / v5 topo - gfp - organellar ras constructs together with pog44 as indicated by the manufacturer 's instructions. after 48 h, exchange into selection media (dmem, 10% fbs, 200 g / ml hygromycin b) initiated death of non - transfected cells. clones derived from single cells were picked and expanded and those positive for organellar ras expression were identified and verified using fluorescence imaging and western blotting. to measure proliferation, cells were cultured at a density of 2000 cells per well in 96-well plates. after 48 h, celltiter 96 aqueous one solution reagent (promega) was added to each well according to the manufacturer 's instructions. after 4 h in culture the cell viability was determined by measuring the absorbance at 490 nm using a multiskan spectrum plate reader (thermo labsystems). values were converted from absorbance to cell number using a standard curve prepared for each experiment. for cisplatin resistance measurements, subconfluent cell cultures were incubated with 30 m cisplatin (cddp ; sigma) for 24 h. cells were lysed on ice using ripa buffer (10 mm tris - cl ph 7.5, 150 mm nacl, 1% triton, 0.1% sds, 1% sodium deoxycholate) lysates were separated by sds page followed by immunoblotting, and finally analyzed with a li - cor odyssey 2.1 system. nih3t3 cells were plated in dmem containing 10% fbs at a density of 5 10 cells/60-mm plate. sixteen hours later the cells were transfected with 1 g of each of activated organellar ras and the following day media was exchanged for dmem, 10% fbs containing 750 g / ml g418. after 14 days the cells were fixed with 0.2% glutaraldehyde, 0.5% formaldehyde in pbs for 10 min on ice and stained with 0.2% crystal violet for 10 min at room temperature. the number of foci 2 mm diameter was counted ; data represent means sem of foci per well from three biological repeats. confluent organellar ras nih3t3 cells were washed with ice - cold pbs and lysed on ice in 50 mm tris - cl (ph 7.2), 500 mm nacl, 10 mm mgcl2, 1% triton x-100, 0.5% sodium deoxycholate and 1:250 protease - inhibitor cocktail (sigma). in order to affinity - purify activated ras from 200 g of lysates, 11 g of gst - rbd k85a fusion protein linked to glutathione sepharose 4b was added to each lysate together with at least a five - fold volume excess of binding buffer (50 mm tris hcl, ph 7.2, 10 mm mgcl2, 0.5 mg / ml bovine serum albumin (bsa), 0.5 mm dithiothreitol and 150 mm nacl) and incubated for 30 min at 4 c. beads were washed twice in binding buffer (without bsa) before boiling in sds sample buffer for 10 min. activated ras in the pull - downs was detected by western blotting with anti - gfp. gst - rbd k85a plasmid was a gift from dr tony burgess, ludwig institute, melbourne, australia. rabbit anti - phospho - akt (ser473), rabbit anti - phospho - mek1/2 (ser217/221), and mouse anti - cleaved parp (asp214) were all from cell signaling technology ; mouse anti - tubulin (sigma) ; mouse anti - tom20 (bd biosciences) ; rabbit anti - gfp ; rabbit anti - eea1. wild type and activated (g12v) gfp - n - ras, gfp - er / golgi - ras (gfp - n - hvr - ala) and gfp - n - ras acceptor constructs lacking the c - terminal hvr have been previously described (laude and prior, 2008). the gfp - n - ras acceptor constructs allow subcloning of membrane targeting motifs c - terminal to the ras g domain separated by a three amino acid ara - linker sequence. 274358), the gm130 golgi targeting domain (aa 784986) and the omp25 mitochondrial targeting domain (aa 170206) from plasmids encoding the relevant proteins : fens_fyve_f : 5-actcgaggtgagctcagcagagaagaggctcct-3 and fens_fyve_r : 5-ctcgaggcccccaccgccacgagaagtccgatcttcatctttgatggagtc-3. gm130_f : 5-ctcgagctgctcacttggcagcccca-3 and gm130_r : 5-gaattcttatataaccatgattttcacctcgtcgttctc-3 ; omp25_f : 5-ctcgaggacatcgaggcgacggagaggcc-3 and omp25_r : 5-gaattccctcagagctgctttcggtatctcacgaaggc-3. the sequenced targeting motifs were subcloned into the n - ras acceptor construct and correct targeting confirmed in hela cells by immunofluorescence microscopy. hela cells were grown at 37 c in dmem (invitrogen) supplemented with 10% heat - inactivated fbs. nih3t3 flp - in cells (invitrogen) were grown in the presence of 100 g / ml zeocin. for the stable expression of compartmentalized ras, nih3t3 flp - in cells were transfected with pef5/frt / v5 topo - gfp - organellar ras constructs together with pog44 as indicated by the manufacturer 's instructions. after 48 h, exchange into selection media (dmem, 10% fbs, 200 g / ml hygromycin b) initiated death of non - transfected cells. clones derived from single cells were picked and expanded and those positive for organellar ras expression were identified and verified using fluorescence imaging and western blotting. to measure proliferation, cells were cultured at a density of 2000 cells per well in 96-well plates. after 48 h, celltiter 96 aqueous one solution reagent (promega) after 4 h in culture the cell viability was determined by measuring the absorbance at 490 nm using a multiskan spectrum plate reader (thermo labsystems). values were converted from absorbance to cell number using a standard curve prepared for each experiment. for cisplatin resistance measurements, subconfluent cell cultures were incubated with 30 m cisplatin (cddp ; sigma) for 24 h. cells were lysed on ice using ripa buffer (10 mm tris - cl ph 7.5, 150 mm nacl, 1% triton, 0.1% sds, 1% sodium deoxycholate) lysates were separated by sds page followed by immunoblotting, and finally analyzed with a li - cor odyssey 2.1 system. nih3t3 cells were plated in dmem containing 10% fbs at a density of 5 10 cells/60-mm plate. sixteen hours later the cells were transfected with 1 g of each of activated organellar ras and the following day media was exchanged for dmem, 10% fbs containing 750 g / ml g418. after 14 days the cells were fixed with 0.2% glutaraldehyde, 0.5% formaldehyde in pbs for 10 min on ice and stained with 0.2% crystal violet for 10 min at room temperature. the number of foci 2 mm diameter was counted ; data represent means sem of foci per well from three biological repeats. confluent organellar ras nih3t3 cells were washed with ice - cold pbs and lysed on ice in 50 mm tris - cl (ph 7.2), 500 mm nacl, 10 mm mgcl2, 1% triton x-100, 0.5% sodium deoxycholate and 1:250 protease - inhibitor cocktail (sigma). in order to affinity - purify activated ras from 200 g of lysates, 11 g of gst - rbd k85a fusion protein linked to glutathione sepharose 4b was added to each lysate together with at least a five - fold volume excess of binding buffer (50 mm tris hcl, ph 7.2, 10 mm mgcl2, 0.5 mg / ml bovine serum albumin (bsa), 0.5 mm dithiothreitol and 150 mm nacl) and incubated for 30 min at 4 c. beads were washed twice in binding buffer (without bsa) before boiling in sds sample buffer for 10 min. activated ras in the pull - downs gst - rbd k85a plasmid was a gift from dr tony burgess, ludwig institute, melbourne, australia. | ras isoforms are membrane bound proteins that differentially localize to the plasma membrane and subcellular compartments within the cell. whilst the cell surface is the main site for ras activity the extent to which intracellular pools contribute to ras function is debated. we have generated ras chimeras targeting ras to the er, golgi, mitochondria and endosomes to compare the capacity of each of these locations to support activity equivalent to normal ras function. we find that all locations are capable of regulating the map kinase and akt pathways. furthermore, whilst endomembranous ras pools show location - specific competence to support proliferation and transformation, golgi - ras is as potent as n - ras. |
despite the complexity of language and learning disorders, individual genes are being defined which appear to influence the development of abilities that are necessary in speech, language, and reading. most of the identified candidate genes involve reading disability, and although the evidence supporting some of these genes is still somewhat tenuous due to small sample sizes and limited replication, most are known to be involved in early development, particularly neuronal migration (galaburda 2005 ; gabel. most of these candidate genes have been associated with several learning and language phenotypes, suggesting that they facilitate learning processes which are basic to learning reading and language. similar pleiotropic effects are seen for several genes that primarily affect autism or language but have also shown effects on reading, including cntnap2 and atp2c2 (vernes. 2008 ; newbury. 2011). however, despite replicated evidence for association of single nucleotide polymorphisms within and around the genes, very few coding mutations have been reported to account for their influence on these disorders. this has led to the hypothesis that mutations affecting reading and related disorders are likely to be in regulatory regions, controlling the quantity rather than quality of the gene product (bates. alterations of gene expression can be caused by mutations in gene promoters and enhancers located near the gene, but mutations in genes that mediate epigenetic controls of gene expression have been found that affect developmental learning disorders. these mutations may be in regions located further from the target gene, making it more difficult to recognize their significance. of all of the genes that have been proposed as candidates for reading disability and language impairment, six genes have been well characterized with respect to their influence on reading and language disorders, the regions within and around the genes that appear to contain causal mutations, and the effects of the putative mutations or risk alleles on gene transcription : dyx1c1, dcdc2, kiaa0319, robo1, and the co - regulated genes mrpl1 and c2orf3. dyx1c1 the 15q21 region was identified as a candidate region for a gene or genes influencing reading disability (rd) through linkage studies (fulker. the dyx1c1 (dyx1-candidate 1) gene was specifically targeted after a translocation t(2;15) (q11;q21) disrupting the previously uncharacterized gene was observed in a family with rd (taipale. since then, the dyx1c1 protein has been found to contain estrogen - receptor - binding sites (massinen. 2009) and knockdown of the gene in embryonic rat brain produces delays in neuronal migration (wang. 2006).sequence analysis of the dyx1c1 coding regions identified a missense mutation in some rd families : rs57809907, 1249g > t, which results in glu417x and truncates the protein by four amino acids (taipale. 2003), but this variant has not been consistently associated with reading disability in other studies (scerri. 2004 ; wigg. another missense mutation, rs17819126 (271g > a, val91ile) has been associated with reading ability (bates. 2010), but analyses of putative effect on protein function by the sift (ng and henikoff 2003) and polyphen (adzhubei. 2010) algorithms indicate that this should be a benign change for the protein (ensembl release 63 : www.ensembl.org). since studies have found association of rd with other snps within the gene, it seems likely that mutations affecting rd are in the regulatory rather than coding regions. a possible candidate is the 3g > a snp rs3743205 in the 5 untranslated region (utr). in the original report by taipale. (2005), the a allele was associated with rd, but subsequent reports found association with the common g allele (scerri. interestingly, a study of rd in chinese children also found strong association with the g allele (lim. studies of transcription factor binding in the promoter region have shown that the a allele shows decreased binding to a repressive transcription factor, resulting in increased dyx1c1 expression (tapia - paez. 2008), leading lim. to hypothesize that the a allele is actually protective compared to the downregulating g allele, and that instances where the a allele appeared to be associated with rd could be secondary to linkage disequilibrium with a second causal variant nearby. two other snps, rs12899331 and rs16787, in the promoter region were also found to be involved in transcription factor binding (tapia - paez. 2008), but these were not found to be associated with rd in later studies (dahdouh. thus, further studies are needed to define the role of particular regulatory regions of dyx1c1 in the cause of rd.there is also evidence for pleiotropic effects of dyx1c1 on short - term memory and mental calculation, a mathematics measure (marino. linkage to the dyx1c1 region was found with speech sound disorder phenotypes (ssd) in one study (smith. further studies are needed to determine whether the linkage signal for ssd was actually related to dyx1c1. dcdc2 initial linkage and association analysis defined the dyx2 locus on chromosome 6p22 (grigorenko. 1999 ; gayan. 1999 ; kaplan. 2002 ; deffenbacher. 2004), and several subsequent studies have reported associations with the dcdc2 (doublecortin-2) gene within the region (newbury. 2011 ; meng. the structure of the gene is analogous to the x - linked dcx gene which is known to be involved in microtubular structure and influences neuronal migration. mutation of the dcx gene produces lissencephaly in males and cortical abnormalities in females (des portes. 1998) ; accordingly, knockdown of dcdc2 produces delays in neuronal migration in embryonic rat brain (meng. 2005b).sequence analysis of coding regions of dcdc2 in rd families has not identified causal mutations ; however, association of rd was reported with a deletion in intron 2, termed bv677278, which appeared to contain transcription factor binding sites (meng. still, the importance of this region in gene regulation has been demonstrated by in vitro studies showing that sequences in the region act as enhancers for dcdc2 expression (meng. furthermore, these differences in gene expression may have a measurable phenotypic effect on brain structure in that bv677278 variants have been associated with differences in gray matter volume in unselected individuals (meda. 2008), so this deletion appears to be an example of a mutation in a regulatory region that affects rd.in addition to influencing rd, snps in dcdc2 have been associated with both hyperactive and inattentive forms of adhd, indicating that this gene can affect both disorders (couto. more recently, evidence has been presented that dcdc2 contributes to the risk for autism in families with both dyslexia and autism (cuccaro. kiaa0319 dcdc2 and kiaa0319 coding regions are separated by only 160 kb and were both included in the candidate region defined by linkage and association analyses that identified the dyx2 locus. association of kiaa0319 with rd phenotypes as well as reading in the normal range has been supported by numerous studies (newbury. although the function of the gene is not clear, knockdown of expression in embryonic rat brain results in delayed neuronal migration (paracchini. 2006), similar to the knockdowns of dyx1c1 and dcdc2 noted above.the snps showing association with rd tend to be located in the 5 utr, the first untranslated exon, and the first intron (elbert. expression of the allele containing the rd - associated snp haplotype in this region of kiaa0319 was shown to be decreased in cell lines from individuals with rd (paracchini. moreover, one associated snp, rs9461045, has been shown to have a regulatory function. reporter assays showed that the risk allele, which was hypothesized to create a binding site for the repressor oct-1, resulted in decreased expression of kiaa0319 in vitro, and knockdown of oct-1 restored expression (dennis. 2009).in recognition of the likely influence of epigenetic mechanisms on kiaa0319 expression in the etiology of rd, regions of acetylated histones were mapped in and around the gene in a neuroblastoma cell line to identify promoter regions (couto. a 2.7-kb acetylated region was found spanning the 5 utr, first exon and first intron of kiaa0319 which corresponded to the location of five snps that had been associated with rd phenotypes in other studies. in addition, snps within or very near the acetylated region have been associated with language impairment phenotypes (newbury. 2011 ; rice. 2009) and linkage to the dcdc2/kiaa0319 region has been reported for ssd (smith. 2005). studies in an unselected population did not show effects on language in the normal range, suggesting that this gene has more of an effect on language impairment (scerri. robo1 linkage analysis of a large family localized rd to a region on chromosome 3 (3p12-q13) which was designated dyx5 (nopola - hemmi. 2001). a translocation within this region, t(3;8)(p12;q11), was found in an individual with rd and it was determined that this disrupted the robo1 gene (hannula - jouppi. this gene is the human homologue of the roundabout gene in drosophila and mice and is known to affect axonal guidance through the midline of the cns and spinal cord. the coding regions of the robo1 gene were sequenced in the original dyx5 family, but no causal mutations were found. association was found with a haplotype of snps in the gene in this family, and transcription of the allele containing the risk haplotype was decreased in lymphoblasts from individuals with rd. the individuals snps were not felt to have a regulatory function since they were also noted in unaffected individuals, pointing to an unknown regulatory mutation in the individuals with the risk haplotype. although subsequent studies have not replicated the association of robo1 snps with rd, linkage has been found with ssd (stein and schick 2004) and snp association has been found with phonological buffer deficits in an unselected population (bates. 2011) indicating that the gene s primary effects could be on language abilities related to rd. mrpl19 and c2orf3 the designation of these two genes as candidates in influencing rd rests on the assumption that the causal mutation is in a regulatory region that is about 34 kb from the genes. the 2p16p12 region was first highlighted by a genome - wide microsatellite linkage study in an extended family (fagerheim. 1999), and subsequent linkage studies replicated these results across the region (petryshen. 2002 ; fisher. 2002 ; francks. 2002 ; kaminen. 2003). snp association studies focused on the 2p12 region, with results indicating a region that did not contain recognizable genes (peyrard - janvid. the transcription products of three nearby genes, flj13391, mrpl19, and c2orf3, were examined to determine if the risk haplotype of snps in that region had an effect on gene expression. there was no effect on the transcription of flj13391, but transcripts of one allele from mrlp19 and c2orf3 were decreased in individuals who carried the risk haplotypes in the adjacent region (as determined by heterozygous snps within the coding regions of the two genes). this suggested that an unknown mutation in the region of snp association has an effect on gene expression of both genes. the mrpl19 protein is a component of the mitochondrial ribosome, but the function of the c2orf3 gene is unknown.overall, there is substantial evidence for involvement of mutations in regulatory regions of the primary candidate genes influencing rd, and several of these genes also affect related language and learning disorders. further investigation of epigenetic mechanisms of gene regulation is likely to be profitable, including elements that may be quite distant from the genes they affect, or factors that regulate more than one gene. the term epigenetics refers to the controls of gene expression that are maintained through somatic cell division (and occasionally in germline cells) but do not involve change in the dna code itself. stable epigenetic controls are applied and subsequently maintained in cell lineages during differentiation and cell proliferation, and reversible epigenetic changes in gene expression can occur in differentiated cells in response to external signals (jaenisch and bird 2003 ; ptashne 2007 ; day and sweatt 2011). the two major methods of epigenetic regulation involve changes in methylation of cytosines in regulatory regions of dna or modification of histone proteins, primarily through acetylation and methylation. methylation of cytosines in regulatory elements or the complexing of dna around nucleosomes can block gene expression, while removal of dna methylation or relaxing of histone complexing can make dna more accessible. methylation often acts on cpg islands or shores, which are regions of cytosine guanine dinucleotide sequences in promoters of genes, thus inhibiting the binding of transcription factors. one or both strands may be methylated, which can fine - tune the degree of expression. in addition, methylation of these regions can recruit histone modifications that also block transcription machinery. in contrast, methylation within the gene exons and introns is correlated with gene expression. dna methylation is mediated by a family of dnmt enzymes which apply and maintain methylation tags (day and sweatt 2011 ; portela and esteller 2010 ; gropman and batshaw 2010). histone modification affects the wrapping of dna around nucleosomes, which are octomers composed of two each of four different histone proteins : h2a, h2b, h3, h4. dna complexing with nucleosomes is part of chromatin compaction into heterochromatin, which generally is less transcriptionally active. each histone protein has multiple sites that are subject to modification (methylation, acetylation, phosphorylation, ubiquination, adp ribosylation, or sumoylation) (kouzarides 2007). specific sites are designated by the histone type and the amino acid number, such that h4k12 designates the 14th amino acid in an h4 protein, which is a lysine (k). these modifications are reversible, mediated by families of enzymes such as histone acetylases (hats), histone deacetylases (hdacs), histone methylases (hmts), histone demethylases (hdms), and so on. code or language that determines when, where, and how much a particular gene is expressed (day and sweatt 2011 ; portela and esteller 2010 ; lee. since epigenetic mechanisms regulate the differential expression of genes in developing tissues, gene mutations that interfere with dna methylation or histone modification may disrupt multiple organ systems. table 1 gives several examples of developmental cognitive disorders caused by mutations in genes that disrupt epigenetic processes resulting in varying degrees of motor, craniofacial, and skeletal problems in addition to their effects on cognitive abilities. other cognitive disorders such as alzheimer disease and huntington disease develop in adulthood through gradual neurodegeneration secondary to deregulated genes.table 1developmental disorders resulting from disruption of epigenetic mechanisms (galaburda 2005)mechanismdiseasegeneeffectconsequencesdna methylationrett syndromemecp2hypermethylation, abnormal mrna splicingtranscription repression or activationfragile x syndromefmr1promoter hypermethylationtranscription repressionprader willi syndrome / angelman syndromedel15q11-q13, ube3aaberrant methylation in imprint control regiontranscription repression or activationimmunodeficiency, centromere instability, facial dysmorphismdnmt3bhypomethylationtranscription activationalzheimer diseasenepcpg island hypomethylationtranscription activationhistone acetylationrubenstein - taybi syndromecbp (hat)reduced histone acetylation, hypertrimethylation of dnatranscription repressioncoffin - lowry syndromersk32hypophosphorylation of site h3s10increased transcription of map kinase genesoculofaciocardio - dentalbcordisruption of hdacstranscription activationhistone methylationsotos syndromensd1decreased methylation of sites h4k20, h3k36transcription activation of multiple geneskleefstra syndromeehmt1decreased histone methylationtranscription activationhuntington diseasehttincreased methylation at site h3k9 and possibly h3k27transcription activationgalaburda (2005) adapted from portela and esteller (2010) ; day and sweatt (2011) ; kelly. (2010) ; and gropman and batshaw (2010) developmental disorders resulting from disruption of epigenetic mechanisms (galaburda 2005) galaburda (2005) adapted from portela and esteller (2010) ; day and sweatt (2011) ; kelly. (2010) ; and gropman and batshaw (2010) while the effects of mutations of genes that affect epigenetic processes can be severe and disrupt multiple systems, other genetic effects on epigenetic modification can be much more circumscribed. the language of methylated dna and specific histone modifications can precisely control gene expression to produce and maintain tissue - specific and region - specific cellular differentiation. once differentiation is completed, the same regulatory mechanisms appear to be involved in the changes in gene expression that result from learning and memory in the hippocampus (day and sweatt 2011). for example, certain types of learning are correlated with specific patterns of histone modification in chromatin of hippocampal cells, e.g., the learning of contextual fear responses in mice is associated with acetylation at h3k9, h3k14, h4k5, h4k8, and h4k12, as well as changes in methylation and phosphorylation at other sites. moreover, loss of acetylation at h4k12 interferes with learning, which is normalized by introduction of an hdac inhibitor which restores actylation at that site (day and sweatt 2011 ; peleg. interference with the machinery that applies histone modifications or dna methylation such as hats, hdacs, hdms, or dnmts also cause learning problems ; for example, mutation of the cbp gene in mice, or blockage of dna methylation through inhibition of dnmts will both interfere with memory and long - term potentiation in the hippocampus (day and sweatt 2011 ; alarcon. 2004 ; lubin. 2008 ; levenson. 2006 ; miller and sweatt 2007). the ehmt gene in humans encodes a histone demethylase and heterozygous deletion of this telomeric gene causes kleefstra syndrome, a condition with severe intellectual disability, dysmorphic features, and behavioral problems such as autistic features, aggression, and bipolar disorder that can change in expression and severity over time (kleefstra. 2009). in drosophila, mutation of the ehmt homologue results in disruption of a jumping reflex and courtship memory. these deficits were also rescued by expression of ehmt in adult flies (kramer. 2011). additional studies of mouse models of alzheimer disease and other neurodegenerative diseases have also shown rescue of learning deficits with treatment by hdacs (fischer. 2007 ; guan. 2009). most recently, there have been several reports of alterations of methylation in autism spectrum disorders. alterations in methylation of cpg islands associated with the oxtr oxytocin receptor gene have been reported in brain tissues of individuals with autism spectrum disorders (gregory. abstracts at the international congress of human genetics / american society of human genetics meeting in montreal in october 2011 reported that identical twins discordant for autism had significantly different genome - wide methylation patterns (wong. 2011), and siblings discordant for autism had differences in 5-hydroxymethylcytosine across exonic sequences. finally, dna methylation was altered in cpg islands associated with the candidate gene shank3 in brain samples from individuals with autism spectrum disorders, resulting in an altered pattern of isoform expression (zhu. the influence of more remote regulatory regions was noted in the downregulation of the chrna7 gene in autism by the prader willi imprinting center at 15q11.213.3 (yasui. mouse models of human epigenetic syndromes, such as those listed in table 1, can show severe phenotypic effects similar to their human counterparts (unless the models are constructed such that the mutations are only expressed in selected tissues) ; however, many of these disorders are caused by null mutations that have a significant effect on function. other models of mutations of genes affecting epigenetic regulation can show much milder changes in hippocampal neurons or dendritic spines (lagali. 2010). it seems possible, then, that less disruptive mutations or mutations of other genes may have much more focused effects on development and thus may be much more analogous to deficits that affect reading and language disorders. thus, while mutations affecting epigenetic mechanisms have not been described in reading disability or language impairments, the role of epigenetic changes in learning and autism and the hints of potential therapy make it especially worthwhile to look for mutations in such genes in individuals with language and learning problems. although candidate genes have been identified for reading disability and language impairment, the snps in these genes appear to account for a small portion of the phenotypic variability. in contrast, fairly substantial heritabilities have been claimed for these disorders, between 0.450.85 depending on population and definitions (gayan and olson 2001 ; hawke. there are several possible explanations for this missing heritability, but one of the primary reasons appears to be inherent in the current studies of snps, particularly in the large panels that are used for genome - wide studies. the snps selected for such panels are generally common in the population, which makes them more informative in comparisons between affected and unaffected individuals, but assessment of individual common snps ignores rare variants which are likely to have more impact, and also ignores epistatic interactions between loci (manolio. there are approaches that enhance the identification of causal genes from genome - wide association studies (gwas) data such as the simultaneous analysis of multiple variants associated with a gene (neale and sham 2004 ; huang. 2011 ; li. 2011) or focus on snps associated with loci which show phenotype - based differences in expression (eqtls or esnps) (innocenti. 2011 ; majewski and pastinen 2011) and next generation sequencing allows the analysis of rare as well as common variants around a gene ; however, sites involved in epigenetic control of gene expression may not be included in the set of loci in gene - based analysis, and the variation in expression of eqtls may be due in part to mutations in epigenetic regions which may be somewhat distant from the gene itself (ernst. the influence of remote regulatory elements is likely to be missing in targeted screening approaches which focus on candidate genes, whether through snp analysis or sequencing. this is due in part to the lack of information on where these regions are located, and initiatives such as the nih epigenomics roadmap program (http://nihroadmap.nih.gov/epigenomics/initiatives.asp) and the international human epigenome consortium (http://www.ihec-epigenomes.org/). these are large collaborative efforts to map regions in the genome that are involved in epigenetic regulation, and the results will assist investigators in identifying regions for evaluation. heritable mutations that influence reading and language disorders could be in the genes that regulate epigenetic processes, analogous to the mutations in hdacs or dnmts, or in genes such as mecp2 or in the mapk signaling pathway (day and sweatt 2011). genome - wide association studies or even targeted snp analysis might be able to detect such mutations, given that the sample size is large enough, the variants are not rare, and the adjacent snps are in linkage disequilibrium. knowledge of the location of epigenetic regions could help prioritize the follow - up of snps in a gwas that otherwise might be ignored because of lack of apparent functional relevance (ernst. 2011), and location information would also guide the placement of snps in a targeted array. sequence analysis would detect rare variants, but until whole genome sequencing of large populations is financially feasible, targeted sequencing studies are also dependent upon the selection of candidate genes and regulatory regions. studies of epigenetic mechanisms in animal models should produce additional candidate genes for examination in cognitive disorders in humans. another approach would be to look for genomic regions of abnormal methylation or histone modification in individuals with specific forms of language or learning disorders. however, epigenetic patterns are likely to be different in different tissues, and histone modifications especially may change over time. fortunately, there are studies which indicate that methylation patterns affecting disorders can be consistent across tissues, such as lymphocyte and brain methylation patterns in individuals with psychiatric disorders, suggesting that lymphocyte tissues can be a good proxy for brain (dempster. an abnormal methylation pattern in a region of dna from human tissues such as lymphocytes or fibroblasts could indicate an epigenetic process that could be pursued further by determination of the effects of that abnormality on gene expression and the impact on learning in animal models. such studies could be valuable in identifying important genes and signaling pathways involved in learning. further genetic studies such as association and sequencing could assess the influence of these new candidates at the population level. conversely, though, lack of a methylation abnormality in proxy tissues would not rule out the involvement of an epigenetic mechanism that is confined to a region of the brain. there are many approaches to the identification of genes that affect quantitative traits such as language and learning disorders, and the most effective will take advantage of simultaneous analysis of genomic and expression analyses (charlesworth. the inclusion of information on epigenetic mechanisms of gene regulation may turn out to be an important consideration in gene identification and possibly even in therapy. of all of the genes that have been proposed as candidates for reading disability and language impairment, six genes have been well characterized with respect to their influence on reading and language disorders, the regions within and around the genes that appear to contain causal mutations, and the effects of the putative mutations or risk alleles on gene transcription : dyx1c1, dcdc2, kiaa0319, robo1, and the co - regulated genes mrpl1 and c2orf3. dyx1c1 the 15q21 region was identified as a candidate region for a gene or genes influencing reading disability (rd) through linkage studies (fulker. the dyx1c1 (dyx1-candidate 1) gene was specifically targeted after a translocation t(2;15) (q11;q21) disrupting the previously uncharacterized gene was observed in a family with rd (taipale. since then, the dyx1c1 protein has been found to contain estrogen - receptor - binding sites (massinen. 2009) and knockdown of the gene in embryonic rat brain produces delays in neuronal migration (wang. 2006).sequence analysis of the dyx1c1 coding regions identified a missense mutation in some rd families : rs57809907, 1249g > t, which results in glu417x and truncates the protein by four amino acids (taipale. 2003), but this variant has not been consistently associated with reading disability in other studies (scerri. 2004 ; marino. 2005 ; meng. 2005a ; dahdouh. 2009 another missense mutation, rs17819126 (271g > a, val91ile) has been associated with reading ability (bates. 2010), but analyses of putative effect on protein function by the sift (ng and henikoff 2003) and polyphen (adzhubei. 2010) algorithms indicate that this should be a benign change for the protein (ensembl release 63 : www.ensembl.org). since studies have found association of rd with other snps within the gene, it seems likely that mutations affecting rd are in the regulatory rather than coding regions. a possible candidate is the 3g > a snp rs3743205 in the 5 untranslated region (utr). in the original report by taipale. (2005), the a allele was associated with rd, but subsequent reports found association with the common g allele (scerri. interestingly, a study of rd in chinese children also found strong association with the g allele (lim. studies of transcription factor binding in the promoter region have shown that the a allele shows decreased binding to a repressive transcription factor, resulting in increased dyx1c1 expression (tapia - paez. 2008), leading lim. to hypothesize that the a allele is actually protective compared to the downregulating g allele, and that instances where the a allele appeared to be associated with rd could be secondary to linkage disequilibrium with a second causal variant nearby two other snps, rs12899331 and rs16787, in the promoter region were also found to be involved in transcription factor binding (tapia - paez. 2008), but these were not found to be associated with rd in later studies (dahdouh. thus, further studies are needed to define the role of particular regulatory regions of dyx1c1 in the cause of rd.there is also evidence for pleiotropic effects of dyx1c1 on short - term memory and mental calculation, a mathematics measure (marino. linkage to the dyx1c1 region was found with speech sound disorder phenotypes (ssd) in one study (smith. 2005) but not in a subsequent study (stein. 2006), which located the ssd region more centromerically. further studies are needed to determine whether the linkage signal for ssd was actually related to dyx1c1. dcdc2 initial linkage and association analysis defined the dyx2 locus on chromosome 6p22 (grigorenko. 1994 ; cardon. 1995 ; fisher. 1999 ; gayan. 1999 ; kaplan. 2002 ; deffenbacher. 2004), and several subsequent studies have reported associations with the dcdc2 (doublecortin-2) gene within the region (newbury. 2011 ; meng. the structure of the gene is analogous to the x - linked dcx gene which is known to be involved in microtubular structure and influences neuronal migration. mutation of the dcx gene produces lissencephaly in males and cortical abnormalities in females (des portes. 1998) ; accordingly, knockdown of dcdc2 produces delays in neuronal migration in embryonic rat brain (meng. 2005b).sequence analysis of coding regions of dcdc2 in rd families has not identified causal mutations ; however, association of rd was reported with a deletion in intron 2, termed bv677278, which appeared to contain transcription factor binding sites (meng. 2008), but other studies have replicated it (brkanac. 2007 ; harold. 2006 ; wilcke. 2009 ; marino. still, the importance of this region in gene regulation has been demonstrated by in vitro studies showing that sequences in the region act as enhancers for dcdc2 expression (meng. furthermore, these differences in gene expression may have a measurable phenotypic effect on brain structure in that bv677278 variants have been associated with differences in gray matter volume in unselected individuals (meda. 2008), so this deletion appears to be an example of a mutation in a regulatory region that affects rd.in addition to influencing rd, snps in dcdc2 have been associated with both hyperactive and inattentive forms of adhd, indicating that this gene can affect both disorders (couto. more recently, evidence has been presented that dcdc2 contributes to the risk for autism in families with both dyslexia and autism (cuccaro. kiaa0319 dcdc2 and kiaa0319 coding regions are separated by only 160 kb and were both included in the candidate region defined by linkage and association analyses that identified the dyx2 locus. association of kiaa0319 with rd phenotypes as well as reading in the normal range has been supported by numerous studies (newbury. 2011 ; dennis. 2009 ; scerri. 2011 ; harold. 2006 ; cope. 2005 ; paracchini. 2006 ; luciano. 2007 ; paracchini. 2008). although the function of the gene is not clear, knockdown of expression in embryonic rat brain results in delayed neuronal migration (paracchini. 2006), similar to the knockdowns of dyx1c1 and dcdc2 noted above.the snps showing association with rd tend to be located in the 5 utr, the first untranslated exon, and the first intron (elbert. expression of the allele containing the rd - associated snp haplotype in this region of kiaa0319 was shown to be decreased in cell lines from individuals with rd (paracchini. moreover, one associated snp, rs9461045, has been shown to have a regulatory function. reporter assays showed that the risk allele, which was hypothesized to create a binding site for the repressor oct-1, resulted in decreased expression of kiaa0319 in vitro, and knockdown of oct-1 restored expression (dennis. 2009).in recognition of the likely influence of epigenetic mechanisms on kiaa0319 expression in the etiology of rd, regions of acetylated histones were mapped in and around the gene in a neuroblastoma cell line to identify promoter regions (couto. a 2.7-kb acetylated region was found spanning the 5 utr, first exon and first intron of kiaa0319 which corresponded to the location of five snps that had been associated with rd phenotypes in other studies. in addition, snps within or very near the acetylated region have been associated with language impairment phenotypes (newbury. 2011 ; rice. 2009) and linkage to the dcdc2/kiaa0319 region has been reported for ssd (smith. 2005). studies in an unselected population did not show effects on language in the normal range, suggesting that this gene has more of an effect on language impairment (scerri. robo1 linkage analysis of a large family localized rd to a region on chromosome 3 (3p12-q13) which was designated dyx5 (nopola - hemmi. 2001). a translocation within this region, t(3;8)(p12;q11), was found in an individual with rd and it was determined that this disrupted the robo1 gene (hannula - jouppi. this gene is the human homologue of the roundabout gene in drosophila and mice and is known to affect axonal guidance through the midline of the cns and spinal cord. the coding regions of the robo1 gene were sequenced in the original dyx5 family, but no causal mutations were found. association was found with a haplotype of snps in the gene in this family, and transcription of the allele containing the risk haplotype was decreased in lymphoblasts from individuals with rd. the individuals snps were not felt to have a regulatory function since they were also noted in unaffected individuals, pointing to an unknown regulatory mutation in the individuals with the risk haplotype. although subsequent studies have not replicated the association of robo1 snps with rd, linkage has been found with ssd (stein and schick 2004) and snp association has been found with phonological buffer deficits in an unselected population (bates. 2011) indicating that the gene s primary effects could be on language abilities related to rd. mrpl19 and c2orf3 the designation of these two genes as candidates in influencing rd rests on the assumption that the causal mutation is in a regulatory region that is about 34 kb from the genes. the 2p16p12 region was first highlighted by a genome - wide microsatellite linkage study in an extended family (fagerheim. 1999), and subsequent linkage studies replicated these results across the region (petryshen. snp association studies focused on the 2p12 region, with results indicating a region that did not contain recognizable genes (peyrard - janvid. the transcription products of three nearby genes, flj13391, mrpl19, and c2orf3, were examined to determine if the risk haplotype of snps in that region had an effect on gene expression. there was no effect on the transcription of flj13391, but transcripts of one allele from mrlp19 and c2orf3 were decreased in individuals who carried the risk haplotypes in the adjacent region (as determined by heterozygous snps within the coding regions of the two genes). this suggested that an unknown mutation in the region of snp association has an effect on gene expression of both genes. the mrpl19 protein is a component of the mitochondrial ribosome, but the function of the c2orf3 gene is unknown.overall, there is substantial evidence for involvement of mutations in regulatory regions of the primary candidate genes influencing rd, and several of these genes also affect related language and learning disorders. further investigation of epigenetic mechanisms of gene regulation is likely to be profitable, including elements that may be quite distant from the genes they affect, or factors that regulate more than one gene. the term epigenetics refers to the controls of gene expression that are maintained through somatic cell division (and occasionally in germline cells) but do not involve change in the dna code itself. stable epigenetic controls are applied and subsequently maintained in cell lineages during differentiation and cell proliferation, and reversible epigenetic changes in gene expression can occur in differentiated cells in response to external signals (jaenisch and bird 2003 ; ptashne 2007 ; day and sweatt 2011). the two major methods of epigenetic regulation involve changes in methylation of cytosines in regulatory regions of dna or modification of histone proteins, primarily through acetylation and methylation. methylation of cytosines in regulatory elements or the complexing of dna around nucleosomes can block gene expression, while removal of dna methylation or relaxing of histone complexing can make dna more accessible. methylation often acts on cpg islands or shores, which are regions of cytosine guanine dinucleotide sequences in promoters of genes, thus inhibiting the binding of transcription factors. one or both strands may be methylated, which can fine - tune the degree of expression. in addition, methylation of these regions can recruit histone modifications that also block transcription machinery. in contrast, methylation within the gene exons and introns is correlated with gene expression. dna methylation is mediated by a family of dnmt enzymes which apply and maintain methylation tags (day and sweatt 2011 ; portela and esteller 2010 ; gropman and batshaw 2010). histone modification affects the wrapping of dna around nucleosomes, which are octomers composed of two each of four different histone proteins : h2a, h2b, h3, h4. dna complexing with nucleosomes is part of chromatin compaction into heterochromatin, which generally is less transcriptionally active. each histone protein has multiple sites that are subject to modification (methylation, acetylation, phosphorylation, ubiquination, adp ribosylation, or sumoylation) (kouzarides 2007). specific sites are designated by the histone type and the amino acid number, such that h4k12 designates the 14th amino acid in an h4 protein, which is a lysine (k). these modifications are reversible, mediated by families of enzymes such as histone acetylases (hats), histone deacetylases (hdacs), histone methylases (hmts), histone demethylases (hdms), and so on. code or language that determines when, where, and how much a particular gene is expressed (day and sweatt 2011 ; portela and esteller 2010 ; lee. since epigenetic mechanisms regulate the differential expression of genes in developing tissues, gene mutations that interfere with dna methylation or histone modification may disrupt multiple organ systems. table 1 gives several examples of developmental cognitive disorders caused by mutations in genes that disrupt epigenetic processes resulting in varying degrees of motor, craniofacial, and skeletal problems in addition to their effects on cognitive abilities. other cognitive disorders such as alzheimer disease and huntington disease develop in adulthood through gradual neurodegeneration secondary to deregulated genes.table 1developmental disorders resulting from disruption of epigenetic mechanisms (galaburda 2005)mechanismdiseasegeneeffectconsequencesdna methylationrett syndromemecp2hypermethylation, abnormal mrna splicingtranscription repression or activationfragile x syndromefmr1promoter hypermethylationtranscription repressionprader willi syndrome / angelman syndromedel15q11-q13, ube3aaberrant methylation in imprint control regiontranscription repression or activationimmunodeficiency, centromere instability, facial dysmorphismdnmt3bhypomethylationtranscription activationalzheimer diseasenepcpg island hypomethylationtranscription activationhistone acetylationrubenstein - taybi syndromecbp (hat)reduced histone acetylation, hypertrimethylation of dnatranscription repressioncoffin - lowry syndromersk32hypophosphorylation of site h3s10increased transcription of map kinase genesoculofaciocardio - dentalbcordisruption of hdacstranscription activationhistone methylationsotos syndromensd1decreased methylation of sites h4k20, h3k36transcription activation of multiple geneskleefstra syndromeehmt1decreased histone methylationtranscription activationhuntington diseasehttincreased methylation at site h3k9 and possibly h3k27transcription activationgalaburda (2005) adapted from portela and esteller (2010) ; day and sweatt (2011) ; kelly. (2010) ; and gropman and batshaw (2010) developmental disorders resulting from disruption of epigenetic mechanisms (galaburda 2005) galaburda (2005) adapted from portela and esteller (2010) ; day and sweatt (2011) ; kelly. (2010) ; and gropman and batshaw (2010) while the effects of mutations of genes that affect epigenetic processes can be severe and disrupt multiple systems, other genetic effects on epigenetic modification can be much more circumscribed. the language of methylated dna and specific histone modifications can precisely control gene expression to produce and maintain tissue - specific and region - specific cellular differentiation. once differentiation is completed, the same regulatory mechanisms appear to be involved in the changes in gene expression that result from learning and memory in the hippocampus (day and sweatt 2011). for example, certain types of learning are correlated with specific patterns of histone modification in chromatin of hippocampal cells, e.g., the learning of contextual fear responses in mice is associated with acetylation at h3k9, h3k14, h4k5, h4k8, and h4k12, as well as changes in methylation and phosphorylation at other sites. moreover, loss of acetylation at h4k12 interferes with learning, which is normalized by introduction of an hdac inhibitor which restores actylation at that site (day and sweatt 2011 ; peleg. interference with the machinery that applies histone modifications or dna methylation such as hats, hdacs, hdms, or dnmts also cause learning problems ; for example, mutation of the cbp gene in mice, or blockage of dna methylation through inhibition of dnmts will both interfere with memory and long - term potentiation in the hippocampus (day and sweatt 2011 ; alarcon. the ehmt gene in humans encodes a histone demethylase and heterozygous deletion of this telomeric gene causes kleefstra syndrome, a condition with severe intellectual disability, dysmorphic features, and behavioral problems such as autistic features, aggression, and bipolar disorder that can change in expression and severity over time (kleefstra. mutation of the ehmt homologue results in disruption of a jumping reflex and courtship memory. these deficits were also rescued by expression of ehmt in adult flies (kramer. 2011). additional studies of mouse models of alzheimer disease and other neurodegenerative diseases have also shown rescue of learning deficits with treatment by hdacs (fischer. 2007 ; guan. 2009). most recently, there have been several reports of alterations of methylation in autism spectrum disorders. alterations in methylation of cpg islands associated with the oxtr oxytocin receptor gene have been reported in brain tissues of individuals with autism spectrum disorders (gregory. abstracts at the international congress of human genetics / american society of human genetics meeting in montreal in october 2011 reported that identical twins discordant for autism had significantly different genome - wide methylation patterns (wong. 2011), and siblings discordant for autism had differences in 5-hydroxymethylcytosine across exonic sequences. finally, dna methylation was altered in cpg islands associated with the candidate gene shank3 in brain samples from individuals with autism spectrum disorders, resulting in an altered pattern of isoform expression (zhu. the influence of more remote regulatory regions was noted in the downregulation of the chrna7 gene in autism by the prader willi imprinting center at 15q11.213.3 (yasui. mouse models of human epigenetic syndromes, such as those listed in table 1, can show severe phenotypic effects similar to their human counterparts (unless the models are constructed such that the mutations are only expressed in selected tissues) ; however, many of these disorders are caused by null mutations that have a significant effect on function. other models of mutations of genes affecting epigenetic regulation can show much milder changes in hippocampal neurons or dendritic spines (lagali. it seems possible, then, that less disruptive mutations or mutations of other genes may have much more focused effects on development and thus may be much more analogous to deficits that affect reading and language disorders. thus, while mutations affecting epigenetic mechanisms have not been described in reading disability or language impairments, the role of epigenetic changes in learning and autism and the hints of potential therapy make it especially worthwhile to look for mutations in such genes in individuals with language and learning problems. although candidate genes have been identified for reading disability and language impairment, the snps in these genes appear to account for a small portion of the phenotypic variability. in contrast, fairly substantial heritabilities have been claimed for these disorders, between 0.450.85 depending on population and definitions (gayan and olson 2001 ; hawke. there are several possible explanations for this missing heritability, but one of the primary reasons appears to be inherent in the current studies of snps, particularly in the large panels that are used for genome - wide studies. the snps selected for such panels are generally common in the population, which makes them more informative in comparisons between affected and unaffected individuals, but assessment of individual common snps ignores rare variants which are likely to have more impact, and also ignores epistatic interactions between loci (manolio. 2009). there are approaches that enhance the identification of causal genes from genome - wide association studies (gwas) data such as the simultaneous analysis of multiple variants associated with a gene (neale and sham 2004 ; huang. 2011 ; li. 2011) or focus on snps associated with loci which show phenotype - based differences in expression (eqtls or esnps) (innocenti. 2011 ; majewski and pastinen 2011) and next generation sequencing allows the analysis of rare as well as common variants around a gene ; however, sites involved in epigenetic control of gene expression may not be included in the set of loci in gene - based analysis, and the variation in expression of eqtls may be due in part to mutations in epigenetic regions which may be somewhat distant from the gene itself (ernst. the influence of remote regulatory elements is likely to be missing in targeted screening approaches which focus on candidate genes, whether through snp analysis or sequencing. this is due in part to the lack of information on where these regions are located, and initiatives such as the nih epigenomics roadmap program (http://nihroadmap.nih.gov/epigenomics/initiatives.asp) and the international human epigenome consortium (http://www.ihec-epigenomes.org/). these are large collaborative efforts to map regions in the genome that are involved in epigenetic regulation, and the results will assist investigators in identifying regions for evaluation. heritable mutations that influence reading and language disorders could be in the genes that regulate epigenetic processes, analogous to the mutations in hdacs or dnmts, or in genes such as mecp2 or in the mapk signaling pathway (day and sweatt 2011). genome - wide association studies or even targeted snp analysis might be able to detect such mutations, given that the sample size is large enough, the variants are not rare, and the adjacent snps are in linkage disequilibrium. knowledge of the location of epigenetic regions could help prioritize the follow - up of snps in a gwas that otherwise might be ignored because of lack of apparent functional relevance (ernst. 2011), and location information would also guide the placement of snps in a targeted array. sequence analysis would detect rare variants, but until whole genome sequencing of large populations is financially feasible, targeted sequencing studies are also dependent upon the selection of candidate genes and regulatory regions. studies of epigenetic mechanisms in animal models should produce additional candidate genes for examination in cognitive disorders in humans. another approach would be to look for genomic regions of abnormal methylation or histone modification in individuals with specific forms of language or learning disorders. however, epigenetic patterns are likely to be different in different tissues, and histone modifications especially may change over time. fortunately, there are studies which indicate that methylation patterns affecting disorders can be consistent across tissues, such as lymphocyte and brain methylation patterns in individuals with psychiatric disorders, suggesting that lymphocyte tissues can be a good proxy for brain (dempster. an abnormal methylation pattern in a region of dna from human tissues such as lymphocytes or fibroblasts could indicate an epigenetic process that could be pursued further by determination of the effects of that abnormality on gene expression and the impact on learning in animal models. such studies could be valuable in identifying important genes and signaling pathways involved in learning. further genetic studies such as association and sequencing could assess the influence of these new candidates at the population level. conversely, though, lack of a methylation abnormality in proxy tissues would not rule out the involvement of an epigenetic mechanism that is confined to a region of the brain. there are many approaches to the identification of genes that affect quantitative traits such as language and learning disorders, and the most effective will take advantage of simultaneous analysis of genomic and expression analyses (charlesworth. the inclusion of information on epigenetic mechanisms of gene regulation may turn out to be an important consideration in gene identification and possibly even in therapy. | language and learning disorders such as reading disability and language impairment are recognized to be subject to substantial genetic influences, but few causal mutations have been identified in the coding regions of candidate genes. association analyses of single nucleotide polymorphisms have suggested the involvement of regulatory regions of these genes, and a few mutations affecting gene expression levels have been identified, indicating that the quantity rather than the quality of the gene product may be most relevant for these disorders. in addition, several of the candidate genes appear to be involved in neuronal migration, confirming the importance of early developmental processes. accordingly, alterations in epigenetic processes such as dna methylation and histone modification are likely to be important in the causes of language and learning disorders based on their functions in gene regulation. epigenetic processes direct the differentiation of cells in early development when neurological pathways are set down, and mutations in genes involved in epigenetic regulation are known to cause cognitive disorders in humans. epigenetic processes also regulate the changes in gene expression in response to learning, and alterations in histone modification are associated with learning and memory deficits in animals. genetic defects in histone modification have been reversed in animals through therapeutic interventions resulting in rescue of these deficits, making it particularly important to investigate their potential contribution to learning disorders in humans. |
the comments offered here assess the scientific foundations of the 3 petitions (carol marcus, michael miller, and mohan doss) to the nuclear regulatory commission (nrc) proposing a change in the use of the linear nonthreshold (lnt) for risk assessment to the hormesis dose response. this assessment includes the scientific and historical foundations of the lnt recommendation by the national academy of sciences (nas) biological effects of atomic radiation (bear) i committee, genetics panel in 1956 for regulatory agencies to adopt linearity at low dose for ionizing radiation risk assessment, how this occurred, and what it means today for nrc regulations. the comments also assess the scientific foundations of hormesis, including how accurately it predicts low - dose effects and how this model compares with other dose response models such as the lnt and threshold models. finally, it will be shown how hormesis could be applied to cancer risk assessment and how this may be used to optimize the health of radiation - exposed workers and the general public. the use of the lnt for radiation - induced mutation originated in 1928 with a publication by the famous physical chemist gilbert lewis in the journal nature. although this specific hypothesis of lewis would not be generally accepted, subsequent research by several students of herman j. muller provided support for a linearity response for gonadal mutation in male fruit flies at very high doses (ie, several hundred thousand - fold greater than normal background). this was the term used throughout the 1930s and 1940s for what would now be called the lnt. the proportionality rule (ie, lnt) became linked to a mechanism in the mid-1930s via the collaboration of leading radiation geneticists and several prominent physicists, yielding the lnt single - hit theory. this early history is described and critiqued in detail by calabrese. during world war ii, the us atomic energy commission (aec) funded research at the university of rochester to determine the shape of the dose response in the low - dose zone. the stern research is central as it was upon these findings that the lnt would be based and accepted by us regulatory agencies. thus, a careful assessment of their research is essential for an evaluation of the 3 petitions to the nrc. calabrese has shown that the interpretations of stern and his manipulations of the publication process led to ideologically based deliberate distortions of the nature of the dose response in the low - dose zone. the history of the lnt and the roles of stern and muller are assessed in detailed by calabrese. these findings reflect documented deceptive actions by muller on multiple occasions in order to ensure acceptance of the lnt. these publications provide a fundamental backdrop for the critical actions of the bear i committee, genetics panel, which is now summarized. substantial research has recently shown that the nas bear i committee, genetics panel misrepresented the research record in its key technical publication in science (june 1956) that recommended the switch from threshold to lnt for risk assessment. this scientific misconduct has now been extensively documented in peer - reviewed publications. as is presented in the paper by calabrese, the panel was extremely concerned that their recommendation to switch to the lnt model be accepted. however, there were very strong misgivings among the panelists that their lnt recommendations would not be accepted if the panel s uncertainties and fundamental scientific disagreements concerning transgenerational genetic risks were made known via their publications to the scientific community and the general public. these fears are documented in the papers by calabrese via letters and other correspondence of panel members. in the 1956 science paper of the panel, it is written that all geneticists on the panel (ie, 12) were challenged to estimate the number of adverse reproductive genetic outcomes that would occur over 10 generations of us residents at a given level of gonadal radiation exposure. all such written documentations are publically available and provide key documentation to support the conclusions of the paper by calabrese. the evidence shows that the estimates of the expert panelists wildly varied, revealing great uncertainty both within and between expert geneticists. such profoundly large inconsistencies and disagreements were disturbing, and a nonscientific ideologically based decision was made to drop the 3 estimates showing the lowest damage., when the 1956 science paper was published, the authors (ie, nas genetics panel) stated that of the 12 geneticists on the panel only 6 took up the challenge and provided estimates. however, we now know that this was not true and can be shown to be a demonstrably false statement. dropping of the 3 lowest genetic damage estimates reduced a significant amount of variation, yet excessive uncertainty still remained. for the remaining 6 estimates, the uncertainly range was 750-fold and was still considered too excessive and was feared this could jeopardize acceptance for the lnt recommendation. thus, the panel then falsified the science paper by stating their range of uncertainty to be only 100-fold. this falsification of the research record would have been discovered if the data had been published. however, the panel formally voted not to make the data public, and therefore, it became impossible to challenge the falsification of the science paper since no panel member revealed these deceptions. finally, there were 3 panel geneticists who refused to provide estimates because the process was excessively uncertain and could not be relied upon. these perspectives were also deliberately omitted as well from the science paper, further misleading the science journal readership. it shows that the key actions of the bear i genetics panel were dishonest, and yet, it was upon their recommendation that the linearity paradigm became accepted, adopted, and implemented within the united states and worldwide. thus, the foundation of the lnt was based on misrepresentations, intending to mislead regulatory agencies and others. in fact, the nrc publication of 1981 addressing cancer risk assessment makes note of the 1956 genetics panel activity, using this deception - based activity as foundational material. as history demonstrates, the genetics panel was successful in their deceptions because of the great authority of the nas and the willingness of the regulatory and scientific communities to accept what they were told without examining the basis for the recommendation. the problem is that it has taken some 6 decades for these deceptions to be revealed. thus, the regulatory process was literally taken hostage by leading radiation geneticists acting via the prestigious us nas much like a highly infectious virus in order to manipulate and direct the actions of regulatory agencies in the united states and elsewhere to their own ideological viewpoint. the bear i genetics panel deliberately refused to provide any documentation to describe the scientific basis for their recommendation that the lnt be adopted by regulatory agencies. newly uncovered documents reveal that this decision was made in order not to show profound disagreements on uncertainty in risk estimation and to focus on the identification of self - serving grant funding opportunities. more specifically, some 6 months after publication of their landmark 1956 report, the bear genetics panel was challenged by a number of distinguished biologists to provide the documentation upon which it based its linearity decision. it should be known that the nas genetics panel had never developed any written basis for the linearity decision. it was simply by proclamation within the panel as seen by a reading of the panel transcripts. now when forced to confront the reality that it had no written basis, the panel decided that it would not provide one. this outrageous and arrogant decision was shared in writing with the president of the nas at the time (dr detlev bronk), thereby making him fully aware of this decision. yet, he would do nothing to reverse it, making him a party to this decision. following the acceptance of lnt, cancer risk assessment would become strongly model driven as is seen in the later biological effects of ionizing radiation (beir) committee reports starting in 1972. once the lnt concept was accepted as a scientific and inaccessible belief, it was transformed into a model - based construct that could not be proven wrong or easily modified. this was the case even after the discovery of dna repair, apoptosis, adaptive response, hormesis, and other new concepts, all of which could profoundly affect the shape of the dose response in the low - dose zone. hormesis, including radiation hormesis, has a long history going back over 100 years. in fact, as early as 1917, ionizing radiation was shown to significantly enhance the lifespan of the insect model, the confused flour beetle, in an extremely well - designed study that has been repeatedly confirmed. thousands of studies have been published over the past several decades on hormesis and show it to be reproducible, generalized, and independent of biological model, agent, end point, and mechanism. in multiple direct head - to - head comparisons, the hormetic model has strikingly outperformed lnt and threshold models for accuracy in low - dose predictions. it is important to note that the many valid hormesis studies not only clearly show the strengths of hormesis but also demonstrate serious flaws in the lnt model and establish that it can not be used as a default, that is, if the lnt can not be shown to provide accurate estimates in so many experimental systems and for a wide range of end points, including those affecting the process of cancer, then it is not possible to rely upon it as a default dose response risk assessment model. although it is widely quoted that a single valid study can discredit a powerful theory, lnt has been shown to be invalid in not one but multiple thousands of peer - reviewed and reproducible studies, affecting a very broad spectrum of biological models and end points, including each key stage of the process of carcinogenesis including tumor formation. with such extensive documentation showing the limitations of the lnt model, it is not scientifically possible to use the lnt as the default model for risk assessment and the basis for regulatory decision making. the lnt model has always been impossible to prove correct, but it could be proven to be incorrect. although the lnt model is being criticized in these comments for its fraudulent origin and integration into us regulatory agencies and its discrediting by a very large number of valid hormesis studies, the proposal that the nrc is considering is to switch to the hormetic dose response model. the nrc should note that a hormesis database was created nearly 20 years ago via funding from multiple sources but principally via the us air force to the university of massachusetts at amherst. this database is being continuously expanded and now there are several different types of hormetic databases which serve differing purposes. in 2005, the hormesis database provides detailed information on each hormetic dose experiment that first passes rigorous evaluative criteria. the findings indicate that hormesis is highly generalizable and is independent of biological model, level of biological organization (ie, cell, organ, and organism), end points measured, inducing agent (eg, chemical class, physical agents such as ionizing radiation, etc), developmental processes, gender, and mechanism. the quantitative features of the hormetic dose response are similar across all of the above - mentioned parameters, suggesting that the hormetic response is constrained by the limits of biological plasticity. also, unlike the lnt model, it can be tested in the observable range and accepted or rejected for any specific experiment. this is a very valuable feature as one does not have to rely on extrapolative modeling but on empirical data. in the early 2000s, the most significant concern with the hormesis model was that it needed to be explained in mechanistic terms. today, this is not a concern and is useful only as a historical note. for example, in 2013, calabrese provided specific mechanisms for 400 different hormetic dose responses, where the response was mediated by a specific receptor and/or cell signaling pathway. no other dose response model has had such a plethora of mechanistic documentation to support and explain it. further, a new hormesis mechanism paper by calabrese is in its final stages of preparation prior to submittal to a journal.. these developments of the past 2 decades have provided information on the occurrence of hormetic dose it provides a sound foundation upon which to build a regulatory program, especially given the fact that its conclusions and predictions are testable. these features make the hormetic dose response a sound choice upon which to base risk assessments upon, including cancer and noncancer end points. these goals can be achieved best at present via the integration of lnt and hormesis models via a model uncertainty methodology. recent papers by calabrese demonstrate that the public health would be optimized at an lnt - based risk of 10, the dose of the hormetic nadir in animal studies. this integration yields the optimal public health response within the context of both defining and minimizing risk model uncertainty, with lnt providing the upper bound and hormesis the lower bound of risks. thus, the nrc should change from an lnt model - based risk assessment as a default to the integrated lnt hormesis model as described by calabrese. | on june 23, 2015, the us nuclear regulatory commission (nrc) issued a formal notice in the federal register that it would consider whether it should amend its standards for protection against radiation regulations from the linear non - threshold (lnt) model of radiation protection to the hormesis model. the present commentary supports this recommendation based on the (1) flawed and deceptive history of the adoption of lnt by the us national academy of sciences (nas) in 1956 ; (2) the documented capacity of hormesis to make more accurate predictions of biological responses for diverse biological end points in the low - dose zone ; (3) the occurrence of extensive hormetic data from the peer - reviewed biomedical literature that revealed hormetic responses are highly generalizable, being independent of biological model, end point measured, inducing agent, level of biological organization, and mechanism ; and (4) the integration of hormesis and lnt models via a model uncertainty methodology that optimizes public health responses at 104. thus, both lnt and hormesis can be integratively used for risk assessment purposes, and this integration defines the so - called regulatory sweet spot. |
cannabinoids are a unique family of terpenophenolic active constituents of cannabis sativa ; 9-tetrahydrocannabinol (thc) is the most relevant member, owing to its psychoactive effects and a wide variety of pharmacological effects. the isolation and characterization of thc allowed for the identification of two distinct cannabinoid receptors (cbrs), named cb1 receptor (cb1r) and cb2 receptor (cb2r), that have been cloned and characterized from mammalian tissues. is abundantly expressed in the central nervous system (cns), with the highest densities in the hippocampus, cerebellum, and striatum. locations outside the brain have also been indicated, including adipose tissue, liver, muscle, the gastrointestinal tract, pancreas, urinary bladder, lung, heart, adrenal gland, testis, uterus, and prostate. in contrast, cb2r has been reported to be essentially limited to the cells associated with the immune system, such as spleen, thymus, and tonsils, but it also has been found in low concentrations in the brain. since the discovery of the cbrs and their endogenous ligands, numerous studies implicate the endocannabinoid system in several physiological and pathological processes, including cancer, appetite, fertility, memory, neuropathic and inflammatory pain, obesity, and neurodegenerative disease. at the present time, cb2r has gained attention as a potential target for immunoregulation. recent advances suggest a role for cb2r within the nervous system, particularly in inflammatory conditions such as neurodegenerative disease (parkinson s disease, alzheimer s disease, huntington s disease, multiple sclerosis, etc.), since cb2r is up - regulated in the brain under these conditions and disease states. this finding is supported by observing the pattern of expression of cb2r during microglia differentiation using an in vitro model of multistep activation. additional data suggest that cb2r - selective agonists show promise for suppressing inflammatory and neuropathic pain states. behavioral, electrophysiological, and neurochemical studies all support a role for cb2r activation in modulating inflammatory nociception. moreover, recent reviews have focused on the evidence for the functional neuronal presence and the emerging role of cb2r in neuropsychiatric disorders. finally, cb2r is overexpressed in several tumor cells, and various in vitro studies and animal models have shown that activation of the cb2r induces apoptosis, inhibits tumor growth, and inhibits neo - angiogenesis. the effectiveness of selective cb2r agonists as neuroprotective and anticancer agents prompted us to report our efforts in this field. our aim was to identify new selective cb2r agonists as potential drugs devoid of the psychotropic side effects associated with cb1r. recently we described the synthesis, binding affinities, and pharmacological characterization of a novel series of 1,8-naphthyridin-2(1h)-one-3-carboxamides of general structure a (figure 1), acting as potent and selective cb2r ligands. the concentration - dependent inhibitory action on human basophils activation and the concentration - dependent decrease of cell viability in jurkat cells shown by one of these derivatives strongly suggest that these compounds possess agonist properties on cb2r. general structure of compounds a. in an effort to develop improved naphthyridine - based cb2r ligands and also to develop structure activity relationships (sars) for both cb1r and cb2r, the present paper describes the synthesis and the pharmacological properties of a number of additional 1,8-naphthyridin-2(1h)-one-3-carboxamide derivatives, 126 (summarized in tables 1 and 2, below), in which the central naphthyridine scaffold has been variously functionalized with different substituents in position 1 or 6. the 4-methylcyclohexyl carboxamide group in position 3 has been selected on the basis of binding results obtained for derivatives a. the new compounds were tested in competitive binding assays toward both human recombinant cb1r and cb2r expressed in hek-293 cells and were found to be selective for cb2r. furthermore, the functional activity of the most representative compounds was determined by a -arrestin 2 recruitment assay using u2os cells co - expressing cb2r and -arr2/gfp as well as with a forskolin - stimulated camp assay, demonstrating that the functionality of tested compounds is controlled by the presence of the substituents at position c-6 of the naphthyridine scaffold. finally, docking studies were performed in order to analyze both the complex of the cb2r in its inactive state (r) with antagonists / inverse agonists and the complex of the cb2r in its activated state (r) with agonists. these studies demonstrated that the difference between the pharmacology of these ligands may be in the ability / inability to block the toggle switch w6.48(258) (1 g+ trans) transition. the synthesis of compounds 126 is depicted in schemes 13. as reported in scheme 1 the n1-alkylation of 1,8-naphthyridine-3-carboxamide 27(19) in anhydrous dmf with the suitable halogenated reagent in the presence of cesium carbonate at 50 c for 12 h afforded the desired compounds 110. the carboxylic acid derivatives 1113 were obtained from the corresponding esters 810 by alkaline hydrolysis followed by acidification. reagents and conditions : (i) cs2co3, rcl or rbr, dmf, 50 c, 12 h, 23%85% ; (ii) naoh aq. 10%. treatment of alcohol derivatives 57 with methanesulfonyl chloride in anhydrous dichloromethane and triethylamine at room temperature for 6 h generated mesylates 2830. exposure of 2830 to tetrabutylammonium fluoride in thf at reflux for 4 h provided derivatives 1416. reagents and conditions : (i) ch2cl2, et3n, mscl, r.t., 6 h, 37%86% ; (ii) tbaf, ch2cl2, reflux, 4 h, 47%49%. 2-aminonicotinaldehyde was treated with bromine in glacial acetic acid at room temperature for 24 h to obtain the corresponding 6-bromo derivative 31, which was refluxed with diethyl malonate and in the presence of piperidine in etoh for 24 h to afford ethyl 6-bromo-1,8-naphthyridin-2(1h)-one-3-carboxylate 32. the reaction of ethyl ester 32 with a cis / trans diastereoisomeric mixture of 4-methylcyclohexylamine in a sealed tube for 24 h at 150 c provided the desired carboxamide 33. n - alkylation of 33 in anhydrous dmf with p - fluorobenzyl chloride or 4-(2-chloroethyl)morpholine in the presence of cesium carbonate afforded the desired 1,8-naphthyridin-2-one derivatives 17 and 18, respectively. to obtain compounds 1926, 6-bromo derivatives 17 or 18 were subjected to a cross - coupling reaction with suitable boronic acids in dioxane under suzuki conditions by generating in situ pd(pph3)4 as the catalyst and aqueous na2co3 (2 m) as the base. each crude mixture was purified by flash chromatography. for compounds 17, 20, and 22, reagents and conditions : (i) br2, acoh, 24 h, r.t., 73% ; (ii) diethyl malonate, piperidine, etoh, reflux, 12 h, 90% ; (iii) 4-methylcyclohexylamine, 150 c, 24 h, 65% ; (iv) cs2co3, p - fluorobenzyl chloride or 4-(2-chloroethyl)morpholine, dmf, 50 c, 12 h, 71%, 92% ; (v) ph3p, pd(oac)2, dioxane, na2co3, suitable boronic acid, 150 c, microwave (200 w, 100 psi, 15 min, under stirring), 4194%. the binding affinities (ki values) of target compounds 126 were evaluated by competitive radioligand displacement assays against the human cb1r and cb2r using [h]cp-55,940 as the radioligand for both receptors. the results are summarized in tables 1 and 2, together with the ki values of previously reported morpholinoethyl and p - fluorobenzyl derivatives a1 and a2, respectively, and reference compounds sr144528 and jwh133. data represent mean values for at least three separate experiments performed in duplicate and are expressed as ki (nm) for cb1r and cb2r binding assays. affinity of compounds for cb1r was evaluated using membranes from hek-293 cells transfected with cb1r and [h]cp-55,940. affinity of compounds for cb2r was evaluated using membranes from hek-293 cells transfected with cb2r and [h]cp-55,940. data represent mean values for at least three separate experiments performed in duplicate and are expressed as ki (nm) for cb1r and cb2r binding assays. affinity of compounds for cb1r was evaluated using membranes from hek-293 cells transfected with cb1r and [h]cp-55,940. affinity of compounds for cb2r was evaluated using membranes from hek-293 cells transfected with cb2r and [h]cp-55,940. first efforts to improve the cb2r affinity and selectivity were focused on the introduction of a large variety of alkyl substituents in position n-1 of the 1,8-naphthyridine nucleus. compounds bearing n - butyl (1) and n - pentyl chains (2) display excellent affinities for cb2r and low affinities at cb1r, so these compounds behave similarly to previously studied compounds a1 and a2. interestingly, compounds 37, characterized by a hydroxyalkyl chain, show no affinity toward cb1r, while their cb2r affinity increases with the elongation of the alkyl chain length. in fact, the replacement of the hydroxyethyl group of 3 with a three - carbon (hydroxypropyl in compound 4), a four - carbon linker (hydroxybutyl in compound 5), or a five - carbon linker (hydroxypentyl in compound 6) progressively increased cb2r affinity (ki values varying from 2096 nm to 3.60 nm). on the contrary, compound 7 possessing a further elongated hydroxyhexyl chain, displays a slight loss in cb2r affinity (ki = 18.0 nm). the replacement of the hydroxy group by a fluorine atom allows to increase affinity for cb2r, but significantly decreases the selectivity toward this receptor (see compounds 15, 16), with the exception of compound 14, which has the highest selectivity obtained for fluoroalkyl compounds (ki(cb1)/ki(cb2) = 743). finally, the esters 810 display a good cb2r affinity and no affinity toward cb1r (ki > 10 000 nm), thus showing an important degree of cb2r selectivity. the corresponding carboxylic acids 1113 show no affinity toward cb1r (ki > 10 000 nm) but also very low cb2r affinity. next, with the aim to investigate the impact on the cb2r affinity and selectivity within this series of 1,8-naphthyridin-2(1h)-one-3-carboxamides, various lipophilic groups were introduced at the position c-6 (table 2). in details, compounds 1726 are characterized by the presence of the common 4-methylcyclohexyl carboxamide moiety at position c-3 together with a p - fluorobenzyl or a morpholinoethyl group at position n-1 of the naphthyridine scaffold, since these groups had been shown to be important for cb2r affinity in our previous studies. all of these 6-substituted analogues (1726) maintain high cb2r affinities (ki 96%. high - resolution mass spectra (hrms) were recorded on a thermo scientific q exactive plus mass spectrometer (thermofisher, breman, germany) equipped with an electrospray ionization source. a solution of 1,8-naphthyridine-3-carboxamide 30 (1.42 g, 5.0 mmol) in anhydrous dmf (20 ml) was treated with cesium carbonate (0.43 g, 14.0 mmol) at room temperature for 1 h. the appropriate reagent (10.0 mmol) was added, and the mixture was stirred for 12 h at 50 c. after cooling, the reaction mixture was evaporated in vacuo, yielding the crude products which were purified by crystallization or flash chromatography. h nmr (cdcl3) : 10.03 and 9.65 (2 m, 1h, nh), 8.88 (s, 1h, ar), 8.73 (dd, j = 4.6 and 1.8 hz, 1h, ar), 8.07 (dd, j = 7.4 and 2.0 hz, 1h, ar), 7.27 (m, 1h, ar), 4.61 (t, j = 7.6 hz, 2h, ch2), 4.26 and 3.95 (2 m, 1h, ch), 1.840.89 (m, 19h, cyclohexyl + ch2 + ch3). c nmr (cdcl3) : 162.16, 162.02, 152.09, 149.66, 140.96, 138.71, 123.47, 119.23, 115.11, 49.84, 45.97, 38.54, 34.21, 33.41, 32.21, 31.38, 30.43, 30.55, 29.90, 29.71, 24.78, 22.31, 21.85, 14.20. hrms - esi : m / z calcd for c20h27n3o2 [m+h ], 342.2182 ; found 342.2171. yield 85% ; crystallized from diisopropyl ether ; ms m / z 355 (m). h nmr (cdcl3) : 10.05 and 9.69 (2 m, 1h, nh), 8.85 (s, 1h, ar), 8.71 (m, 1h, ar), 8.06 (m, 1h, ar), 7.27 (m, 1h, ar), 4.58 (t, j = 7.3 hz, 2h, ch2), 4.25 and 3.96 (2 m, 1h, ch), 1.840.92 (m, 21h, cyclohexyl + ch2 + ch3). c nmr (cdcl3) : 162.34, 162.01, 152.15, 149.80, 141.79, 138.65, 123.36, 119.15, 115.21, 49.63, 45.49, 38.93, 34.22, 33.08, 32.15, 31.87, 30.55, 30.48, 29.91, 29.66, 24.23, 23.61, 22.14, 21.38, 14.35. hrms - esi : m / z calcd for c21h29n3o2 [m+h ], 356.2338 ; found 356.2326. h nmr (dmso) : 10.00 and 9.62 (2 m, 1h, nh), 8.88 (s, 1h, ar), 8.78 (m, 1h, ar), 8.07 (m, 1h, ar), 7.44 (m, 1h, ar), 4.62 (m, 2h, ch2), 4.26 and 3.95 (2 m, 1h, ch), 4.17 (exchangeable proton, 1h, oh), 3.67 (m, 2h, ch2), 1.840.89 (m, 12h, cyclohexyl + ch3). c nmr (cdcl3) : 162.45, 162.07, 152.25, 149.34, 141.89, 138.64, 123.23, 119.21, 115.22, 61.71, 49.55, 45.41, 41.72, 34.45, 33.72, 32.42, 31.73, 30.42, 29.96, 22.15, 21.38. hrms - esi : m / z calcd for c18h23n3o3 [m+h ], 330.1818 ; found 330.1807. h nmr (dmso) : 10.00 and 9.62 (2 m, 1h, nh), 8.88 (s, 1h, ar), 8.78 (m, 1h, ar), 8.07 (m, 1h, ar), 7.44 (m, 1h, ar), 4.55 (m, 2h, ch2), 4.26 and 3.95 (2 m, 1h, ch), 4.19 (exchangeable proton, 1h, oh), 3.52 (m, 2h, ch2), 1.840.89 (m, 14h, cyclohexyl + ch2 + ch3). c nmr (cdcl3) : 162.71, 162.12, 152.11, 149.34, 141.65, 138.64, 123.23, 119.21, 115.12, 62.07, 49.00, 45.91, 41.46, 34.15, 33.28, 32.12, 31.30, 30.63, 30.54, 29.44, 22.14, 21.26. hrms - esi : m / z calcd for c19h25n3o3 [m+h ], 344.1974 ; found 344.1963. h nmr (dmso) : 10.01 and 9.61 (2 m, 1h, nh), 8.81 (s, 1h, ar), 8.66 (dd, j = 4.4 and 1.7 hz, 1h, ar), 8.04 (dd, j = 7.2 and 2.0 hz, 1h, ar), 7.24 (m, 1h, ar), 4.55 (m, 2h, ch2), 4.28 and 3.89 (2 m, 1h, ch), 4.18 (exchangeable proton, 1h, oh), 3.73 (m, 2h, ch2), 1.840.89 (m, 16h, cyclohexyl + ch2 + ch3). c nmr (cdcl3) : 162.54, 162.12, 151.97, 149.64, 141.87, 138.81, 123.09, 119.27, 115.12, 62.56, 49.12, 45.79, 41.14, 34.33, 33.51, 32.12, 31.11, 30.44, 29.43, 29.86, 25.75, 22.37, 21.22. hrms - esi : m / z calcd for c20h27n3o3 [m+h ], 358.2131 ; found 358.2120. h nmr (cdcl3) : 10.03 and 9.64 (2 m, 1h, nh) ; 8.87 (s, 1h, ar) ; 8.72 (m, 1h, ar) ; 8.08 (m, 1h, ar) ; 7.28 (m, 1h, ar) ; 4.61 (m, 2h, ch2) ; 4.27 and 3.89 (2 m, 1h, ch) ; 4.15 (exchangeable proton, 1h, oh) 3.69 (m, 2h, ch2) ; 1.820.92 (m, 18h, cyclohexyl + ch2 + ch3). c nmr (cdcl3) : 162.21, 162.04, 152.2, 149.58, 142.06, 138.34, 123.05, 119.37, 115.22, 62.68, 49.35, 45.92, 40.89, 34.21, 33.12, 32.33, 31.13, 30.46, 29.92, 29.78, 27.79, 25.20, 22.26, 21.35. hrms - esi : m / z calcd for c21h29n3o3 [m+h ], 372.2287 ; found 372.2275. h nmr (cdcl3) : 10.00 and 9.65 (2 m, 1h, nh), 8.89 (s, 1h, ar), 8.74 (m, 1h, ar), 8.09 (m, 1h, ar), 7.29 (m, 1h, ar), 4.62 (m, 2h, ch2), 4.26 and 3.89 (2 m, 1h, ch), 4.16 (exchangeable proton, 1h, oh), 3.69 (m, 2h, ch2), 1.830.96 (m, 20h, cyclohexyl + ch2) + ch3). c nmr (cdcl3) : 162.32, 162.25, 152.00, 149.65, 141.48, 138.50, 123.44, 119.21, 115.10, 62.72, 49.40, 45.92, 40.90, 34.19, 33.12, 32.33, 31.43, 30.47, 29.95, 29.79, 27.68, 27.61, 25.49, 22.35, 21.48. hrms - esi : m / z calcd for c22h31n3o3 [m+h ], 386.2444 ; found 386.2430. h nmr (cdcl3) : 10.01 and 9.62 (2 m, 1h, nh) ; 8.90 (s, 1h, ar) ; 8.71 (dd, j = 4.6 and 1.8 hz, 1h, ar) ; 8.08 (dd, j = 7.4 and 2.0 hz, 1h, ar) ; 7.27 (m, 1h, ar) ; 4.90 (m, 2h, ch2) ; 4.14 (m, 2h, ch2) ; 4.25 and 3.97 (2 m, 1h, ch) ; 2.80 (m, 2h, ch2) ; 1.840.89 (m, 15h, cyclohexyl + ch3). c nmr (cdcl3) : 173.69, 162.34, 162.18, 152.28, 149.54, 141.78, 138.58, 123.37, 119.29, 115.39, 61.47, 49.37, 45.79, 40.95, 34.26, 33.36, 33.19, 32.52, 31.43, 30.85, 30.45, 29.78, 22.23, 21.16, 15.67. hrms - esi : m / z calcd for c21h27n3o4 [m+h ], 386.2080 ; found 386.2066. h nmr (cdcl3) : 10.04 and 9.67 (2 m, 1h, nh) ; 8.89 (s, 1h, ar) ; 8.70 (dd, j = 4.6 and 1.8 hz, 1h, ar) ; 8.10 (dd, j = 7.4 and 2.0 hz, 1h, ar) ; 7.26 (m, 1h, ar) ; 4.69 (m, 2h, ch2) ; 4.15 (m, 2h, ch2) ; 4.28 and 3.95 (2 m, 1h, ch) ; 2.45 (m, 2h, ch2) ; 2.15 (m, 2h, ch2) ; 1.840.89 (m, 15h, cyclohexyl + ch3). c nmr (cdcl3) : 173.59, 162.34, 162.21, 152.34, 149.56, 142.09, 138.77, 123.41, 119.21, 115.31, 61.43, 49.88, 45.82, 40.89, 34.22, 33.31, 32.42, 31.37, 31.43, 30.44, 29.87, 23.35, 22.13, 21.46, 15.63. hrms - esi : m / z calcd for c22h29n3o4 [m+h ], 400.2236 ; found 400.2224. h nmr (cdcl3) : 10.03 and 9.66 (2 m, 1h, nh) ; 8.88 (s, 1h, ar) ; 8.71 (dd, j = 4.6 and 1.8 hz, 1h, ar) ; 8.09 (dd, j = 7.4 and 2.0 hz, 1h, ar) ; 7.27 (m, 1h, ar) ; 4.63 (m, 2h, ch2) ; 4.28 and 3.95 (2 m, 1h, ch) ; 3.68 (1s, 3h, ch3) ; 2.45 (m, 2h, ch2) ; 1.820.83 (m, 16h, cyclohexyl + ch2 + ch3). c nmr (cdcl3) : 173.61, 162.42, 162.23, 152.55, 149.36, 141.39, 138.87, 123.24, 119.21, 115.34, 55.82, 49.97, 45.69, 40.39, 34.27, 33.28, 33.19, 32.12, 31.43, 30.95, 30.47, 29.86, 27.41, 23.35, 22.31, 21.46. hrms - esi : m / z calcd for c22h29n3o4 [m+h ], 400.2236 ; found 400.2223. a mixture of 0.21 mmol of ester derivatives 810 in 15.0 ml of naoh 10% was heated at 110 c for 5 h. after cooling, the reaction mixture was treated with water and then with concentrated hcl until ph 23. the precipitate formed was filtered and treated with et2o to give the acid derivatives 1113. h nmr (dmso) : 12.55 (s, 1h, oh) ; 9.83 and 9.68 (2 m, 1h, nh) ; 8.86 (s, 1h, ar) ; 8.76 (m, 1h, ar) ; 8.48 (m, 1h, ar) ; 7.42 (m, 1h, ar) ; 4.68 (m, 2h, ch2) ; 4.25 and 3.97 (2 m, 1h, ch) ; 2.90 (m, 2h, ch2) ; 1.860.90 (m, 12h, cyclohexyl + ch3). c nmr (dmso) : 177.83, 161.48, 161.22, 150.42, 148.45, 142.11, 138.20, 122.44, 117.23, 113.13, 47.68, 44.97, 41.74, 37.80, 34.12, 33.31, 32.42, 31.63, 29.76, 29.25, 22.31, 21.36. hrms - esi : m / z calcd for c19h23n3o4 [m+h ], 358.1767 ; found 358.1756. h nmr (dmso) : 12.06 (s, 1h, oh) ; 9.96 and 9.68 (2 m, 1h, nh) ; 8.90 (s, 1h, ar) ; 8.78 (m, 1h, ar) ; 8.48 (m, 1h, ar) ; 7.44 (m, 1h, ar) ; 4.54 (m, 2h, ch2) ; 4.24 and 3.95 (2 m, 1h, ch) ; 2.49 (m, 2h, ch2) ; 1.940.92 (m, 14h, cyclohexyl + ch2 + ch3). c nmr (dmso) : 177.88, 161.53, 161.36, 150.46, 148.55, 142.21, 138.22, 122.24, 117.28, 113.16, 47.72, 44.95, 43.12, 36.92, 34.15, 33.12, 32.34, 31.33, 29.65, 29.19, 23.79, 22.32, 21.34. hrms - esi : m / z calcd for c20h25n3o4 [m+h ], 372.1923 ; found 372.1912. h nmr (dmso) : 12.05 (s, 1h, oh) ; 9.94 and 9.58 (2 m, 1h, nh) ; 8.90 (s, 1h, ar) ; 8.80 (dd, j = 4.6 and 1.8 hz, 1h, ar) ; 8.49 (dd, j = 7.4 and 2.0 hz, 1h, ar) ; 7.45 (m, 1h, ar) ; 4.49 (m, 2h, ch2) ; 4.12 and 3.85 (2 m, 1h, ch) ; 2.29 (m, 2h, ch2) ; 1.970.88 (m, 16h, cyclohexyl + 2ch2 + ch3). c nmr (dmso) : 177.89, 161.42, 161.21, 150.40, 148.68, 142.00, 138.00, 122.43, 117.23, 113.19, 47.68, 44.97, 41.74, 37.82, 34.35, 33.21, 32.20, 31.33, 29.89, 29.43, 27.85, 24.09, 22.31, 21.46. hrms - esi : m / z calcd for c21h27n3o4 [m+h ], 386.2080 ; found 386.2068. triethylamine (0.82 ml,0.526 g, 5.89 mmol) was added at 0 c to a solution of suitable alcohol 57 (0.98 mmol) and methanesulfonyl chloride (0.23 ml, 0.34 g, 2.94 mmol) in anhydrous dichloromethane (8.0 ml), and the mixture was stirred for 6 h at ambient temperature. after addition of ethyl acetate (30 ml), the mixture was washed with water (20.0 ml) and then with brine (20.0 ml). the organic layer was dried over mgso4 and evaporated to dryness to give a residue which was purified by flash column chromatography. h nmr (cdcl3) : 9.98 and 9.63 (2 m, 1h, nh) ; 8.83 (s, 1h, ar) ; 8.67 (m, 1h, ar) ; 8.08 (m, 1h, ar) ; 7.26 (m, 1h, ar) ; 4.61 (m, 2h, ch2) ; 4.30 (m, 2h, ch2) ; 4.28 and 3.89 (2 m, 1h, ch) ; 3.01(s, 3h, ch3) ; 1.880.87 (m, 16h, cyclohexyl + ch2 + ch3). c nmr (cdcl3) : 162.73, 161.96, 152.22, 149.78, 142.00, 138.69, 123.34, 119.35, 115.11, 69.94, 49.03, 45.83, 41.21, 37.60, 34.14, 33.21, 32.25, 31.36, 30.45, 29.88, 26.84, 24.29, 22.50, 21.87. h nmr (cdcl3) : 9.99 and 9.61 (2 m, 1h, nh) ; 8.86 (s, 1h, ar) ; 8.70 (m, 1h, ar) ; 8.07 (m, 1h, ar) ; 7.26 (m, 1h, ar) ; 4.57 (m, 2h, ch2) ; 4.29 (m, 2h, ch2) ; 4.18 and 3.89 (2 m, 1h, ch) ; 3.00(s, 3h, ch3) ; 1.970.89 (m, 18h, cyclohexyl + ch2 + ch3). c nmr (cdcl3) : 162.73, 162.05, 152.20, 149.78, 142.00, 138.62, 123.41, 119.24, 115.13, 70.05, 49.05, 45.90, 41.71, 37.64, 34.18, 33.25, 32.27, 31.32, 30.48, 29.86, 29.04, 27.44, 23.29, 22.51, 21.79. h nmr (cdcl3) : 9.96 and 9.63 (2 m, 1h, nh) ; 8.82 (s, 1h, ar) ; 8.65 (m, 1h, ar) ; 8.01 (m, 1h, ar) ; 7.20 (m, 1h, ar) ; 4.50 (m, 2h, ch2) ; 4.28 (m, 2h, ch2) ; 4.17 and 3.89 (2 m, 1h, ch) ; 2.96 (s, 3h, ch3) ; 1.990.76 (m, 20h, cyclohexyl + ch2 + ch3). c nmr (cdcl3) : 162.65, 162.07, 152.13, 149.82, 141.80, 138.53, 123.41, 119.12, 115.09, 70.21, 49.02, 45.92, 41.88, 37.64, 34.18, 33.21, 32.49, 31.25, 30.45, 29.81, 29.29, 27.84, 26.68, 25.42, 22.49, 21.72. to a solution of suitable mesylate 2830 (0.6 mmol) in anhydrous thf (10 ml) was added tetrabutylammonium fluoride (1.00 n) in thf (0.35 ml, 0.314 g, 1.2 mmol), and the mixture was refluxed for 4 h. after cooling, the solvent was removed under vacuum, and the residue was solubilized in chloroform (10 ml), washed with water (20 ml), and then washed with brine (20 ml). the organic layer was dried over mgso4 and evaporated to dryness to give crude product which was purified by flash column chromatography. h nmr (cdcl3) : 10.01 and 9.62 (2 m, 1h, nh) ; 8.88 (s, 1h, ar) ; 8.71 (m, 1h, ar) ; 8.08 (m, 1h, ar) ; 7.28 (m, 1h, ar) ; 4.66 (m, 2h, ch2) ; 4.42 (m, 2h, ch2) ; 4.26 and 3.93 (2 m, 1h, ch) ; 1.920.91 (m, 16h, cyclohexyl + ch2 + ch3). c nmr (cdcl3) : 162.76, 162.06, 152.15, 149.91, 141.89, 138.59, 123.47, 119.19, 115.13, 85.61, 82.33, 49.07, 45.94, 41.59, 34.20, 33.27, 32.29, 31.34, 30.48, 29.88, 28.44, 24.23, 22.52, 21.78. hrms - esi : m / z calcd for c20h26fn3o2 [m+h ], 360.2087 ; found 360.2076. h nmr (cdcl3) : 10.03 and 9.64 (2 m, 1h, nh) ; 8.88 (s, 1h, ar) ; 8.71 (m, 1h, ar) ; 8.09 (m, 1h, ar) ; 7.26 (m, 1h, ar) ; 4.59 (m, 2h, ch2) ; 4.37 (m, 2h, ch2) ; 4.28 and 3.95 (2 m, 1h, ch) ; 1.820.92 (m, 18h, cyclohexyl + ch2 + ch3). c nmr (cdcl3) : 162.77, 162.11, 152.15, 149.95, 141.84, 138.59, 123.42, 119.17, 115.15, 85.82, 82.55, 49.73, 45.96, 41.92, 34.21, 33.27, 32.30, 31.24, 30.62, 30.44, 29.88, 27.76, 24.23, 22.72, 21.76. hrms - esi : m / z calcd for c21h28fn3o2 [m+h ], 374.2244 ; found 374.2232. h nmr (cdcl3) : 10.03 and 9.65 (2 m, 1h, nh) ; 8.87 (s, 1h, ar) ; 8.71 (m, 1h, ar) ; 8.08 (m, 1h, ar) ; 7.28 (m, 1h, ar) ; 4.59 (m, 2h, ch2) ; 4.34 (m, 2h, ch2) ; 4.27 and 3.93 (2 m, 1h, ch) ; 1.810.91 c nmr (cdcl3) : 162.77, 162.15, 152.15, 149.95, 141.90, 138.55, 123.42, 119.10, 115.11, 85.93, 82.65, 49.05, 45.968 42.02, 34.21, 33.27, 32.30, 31.24, 30.78, 30.48, 29.90, 28.06, 26.97, 25.26, 22.72, 21.76. hrms - esi : m / z calcd for c22h30fn3o2 [m+h ], 388.2400 ; found 388.2387. to a stirred solution of 2-aminopyridine-3-carboxaldehyde (0.40 g, 3.30 mmol) in 15 ml of glacial acetic acid was added bromine (0.16 ml, 3.16 mmol), and the reaction mixture was stirred at room temperature for 24 h. the precipitate obtained was filtered off and washed with ether. the filter cake was poured into water and treated with solid naoh until ph 78, and the mixture was extracted with dichloromethane. the organic layer was dried with mgso4 and evaporated to dryness under reduced pressure. the crude solid was purified by crystallization in acetonitrile to give 31 (0.48 g, 73%) : mp 147150 c ; ms m / z 199 (m). h nmr (dmso) : 9.83 (s, 1h, cho), 8.33 (s, 1h, ar) ; 8.27 (s,1h, ar) ; 5.27 (br, 2h, nh2). to a solution of 31 (1.50 g, 7.5 mmol) in ethanol (20 ml) were added diethyl malonate (1.80 g, 11.25 mmol) and 0.21 ml of piperidine (0.182 g, 2.14 mmol), and the mixture was stirred under reflux for 20 h. after cooling, the solid obtained was filtered, washed with ethanol, and dried. the crude product was used without further purifications (1.99 g, 90%) : mp 200203 c ; ms m / z 296 (m). h nmr (dmso) : 12.64 (br, 1h, nh) ; 8.69 (d, j = 2.2 hz, 1h, ar) ; 8.56 (d, j = 2.2 hz, 1h, ar) ; 8.45 (s, 1h, ar) ; 4.28 (q, j = 7.3 hz, 2h, ch2) ; 1.31 (t, j = 7.1 hz, 3h, ch3). a mixture of 4 (0.40 g, 1.35 mmol) and 4-methylcyclohexylamine (0.76 g, 6.75 mmol) was heated in a sealed tube at 150 c for 24 h. after cooling, the reaction mixture was treated with diethyl ether to give a solid residue which was collected by filtration. the product was crystallized from ethyl acetate (0.32 g, 65%) : ms m / z 363 (m). h nmr (dmso) : 12.09 (br, 1h, nh) ; 10.06 and 9.57 (2 m, 1h, nh) ; 8.738.83 (m, 3h, ar) ; 4.12 and 3.78 (2 m, 1h, ch) ; 1.900.89 (m, 12h, cyclohexyl + ch3). a solution of 6-bromo - n-(4-methylcyclo - hexyl)-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamide 33 (1.42 g, 5.0 mmol) in anhydrous dmf (20 ml) was treated with cesium carbonate (0.43 g, 14.0 mmol) at room temperature for 1 h. the suitable chloride (10 mmol) was added, and the mixture was stirred for 12 h at 50 c. after cooling, the reaction mixture was evaporated in vacuo, yielding the crude products which were purified by flash chromatography. purified by flash chromatography (toluene / ethyl acetate 14:1 and 1% of acetic acid). h nmr (cdcl3) : 9.92 and 9.52 (2 m, 1h, nh) ; 8.75 (m, 2h, ar) ; 8.20 (s, 1h, ar) ; 7.52 (m, 2h, ar) ; 7.03 (m, 2h, ar) ; 5.73 (s, 2h, ch2), 4.26 and 3.95 (2 m, 1h, ch) ; 1.800.91(m, 12h, cyclohexyl + ch3). c nmr (cdcl3) : 164.39, 162.75, 161.82, 152.57, 151.49, 142.35, 140.92, 138.96, 129.12, 124.75, 116.33, 115.28, 114.16, 49.47, 46.16, 44.88, 34.21, 33.25, 32.20, 31.28, 30.54, 29.86, 22.56, 21.80. hrms - esi : m / z calcd for c23h23brfn3o2 [m+h ], 472.1036 ; found 472.1027. compounds 17-trans and 17-cis were obtained from derivative 17 by flash chromatography on a silica gel (toluene / ethyl acetate 14:1 and 1% of acetic acid). yield 17% ; ms 471 m / z (m) ; mp 163165 c. h nmr (cdcl3) : 9.52 (m, 1h, nh) ; 8.75 (m, 2h, ar) ; 8.20 (s, 1h, ar) ; 7.45 (m, 2h, ar) ; 7.02 (m, 2h, ar) ; 5.72 (s, 2h, ch2), 3.95 (m, 1h, ch) ; 1.790.91 (m, 12h, cyclohexyl + ch3). c nmr (cdcl3) : 164.39, 162.75, 161.82, 152.57, 151.49, 142.35, 140.92, 138.96, 129.12, 124.75, 116.33, 115.28, 114.16, 46.16, 44.88, 33.25, 31.28, 29.86, 21.80. yield 8% ; ms 471 m / z (m) ; mp 158160 c. h nmr (cdcl3) : 9.92 (m, 1h, nh) ; 8.75 (m, 2h, ar) ; 8.20 (s, 1h, ar) ; 7.45 (m, 2h, ar) ; 7.02 (m, 2h, ar) ; 5.72 (s, 2h, ch2), 4.26 (m, 1h, ch) ; 1.790.93 (m, 12h, cyclohexyl + ch3). c nmr (cdcl3) : 164.39, 162.75, 161.82, 152.57, 151.49, 142.35, 140.92, 138.96, 129.12, 124.75, 116.33, 115.28, 114.16, 49.47, 44.88, 34.21, 32.20, 30.54, 22.56. yield 92% (crystallized by acetonitrile) ; ms 476 m / z (m). h nmr (cdcl3) : 9.97 and 9.57 (2 m, 1h, nh) ; 8.75 (m, 2h, ar) ; 8.19 (s, 1h, ar) ; 4.73 (t, j = 6.8 hz, 2h, ch2), 4.24 and 3.96 (2 m, 1h, ch) ; 3.51 (m, 4h, morpholine), 2.57 (m, 6h, morpholine + ch2) ; 1.870.95 (m, 12h, cyclohexyl + ch3). c nmr (cdcl3) : 162.45, 161.52, 152.43, 148.38, 141.11, 139.93, 124.45, 116.31, 114.26, 67.24, 56.12, 54.12, 49.13, 45.89, 39.30, 34.16, 33.21, 32.25, 31.34, 30.41, 29.90, 22.52, 21.91. hrms - esi : m / z calcd for c22h29brn4o3 [m+h ], 477.1501 ; found 477.1491. a mixture of 50 mg of ph3p (0.20 mmol) and 10 mg of pd(oac)2 (0.04 mmol) in dioxane (1.0 ml) was stirred under n2 for 10 min. then the approriate 6-bromo derivative 17 or 18 (0.41 mmol) in 1 ml of meoh, 0.85 ml of na2co3 (10%), and suitable boronic acid (0.82 mmol) were added. the mixture was heated by microwave radiation (cem) at 150 c for 10 min (power 200 w, pressure 100 psi, stirring on). the reaction mixture was then cooled to room temperature, treated with water, and extracted with dichloromethane. the combined organic layers were washed with brine, dried over anhydrous sodium sulfate, and evaporated to dryness to obtain a residue which was purified by flash chromatography on silica gel. h nmr (dmso) : 10.02 and 9.61 (2 m, 1h, nh) ; 8.92 (m, 2h, ar) ; 8.18 (s, 1h, ar) ; 7.55 (m, 4h, ar) ; 7.05 (m, 4h, ar) ; 5.82 (s, 2h, ch2), 3.95 and 4.26 (2 m, 1h, ch) ; 3.88 (s, 3h, ch3) ; 1.600.92 (m, 12h, cyclohexyl + ch3). c nmr (cdcl3) : 164.39, 162.75, 162.12, 161.09, 150.99, 142.35, 140.87, 134.66, 133.80, 129.12, 128.89, 128.13, 125.76, 124.75, 116.33, 115.28, 115.07, 55.78, 49.47, 46.16, 44.88, 34.21, 33.25, 32.20, 31.28, 30.54, 29.86, 22.56, 21.80. hrms - esi : m / z calcd for c30h30fn3o3 [m+h ], 500.2349 ; found 500.2337. h nmr (cdcl3) : 9.98 and 9.47 (2 m, 1h, nh) ; 8.95 (m, 2h, ar) ; 8.21 (s, 1h, ar) ; 7.51 (m, 3h, ar) ; 7.19 (m, 2h, ar) ; 6.99 (m, 2h, ar) ; 5.80 (s, 2h, ch2), 4.26 and 3.94 (2 m, 1h, ch) ; 1.820.95 (m, 12h, cyclohexyl + ch3). c nmr (cdcl3) : 164.39, 162.75, 162.12, 150.90, 142.47, 140.56, 138.85, 134.66, 133.26, 128.86, 127.92, 127.63, 125.56, 124.55, 122.8, 116.33, 115.28, 49.47, 46.16, 44.88, 34.21, 33.25, 32.20, 31.28, 30.54, 29.86, 22.56, 21.80. hrms - esi : m / z calcd for c27h26fn3o2s [m+h ], 476.1808 ; found 476.1797. compounds 20-trans and 20-cis were obtained from derivative 20 by flash chromatography on a silica gel (toluene / ethyl acetate 8:1). yield 24% ; ms 475 m / z (m) ; mp 169171 c. h nmr (cdcl3) : 9.47 (m, 1h, nh) ; 8.97 (m, 2h, ar) ; 8.22 (s, 1h, ar) ; 7.46 (m, 3h, ar) ; 7.19 (m, 2h, ar) ; 6.96 (m, 2h, ar) ; 5.79 (s, 2h, ch2), 3.97 (m, 1h, ch) ; 1.800.92 (m, 12h, cyclohexyl + ch3). c nmr (cdcl3) : 164.39, 162.75, 162.12, 150.90, 142.47, 140.56, 138.85, 134.66, 133.26, 128.86, 127.92, 127.63, 125.56, 124.55, 122.8, 116.33, 115.28, 55.78, 46.16, 44.88, 33.25, 31.28, 29.86, 21.80. yield 21% ; ms 475 m / z (m) ; mp 163165 c. h nmr (cdcl3) : 9.47 (m, 1h, nh) ; 8.97 (m, 2h, ar) ; 8.22 (s, 1h, ar) ; 7.46 (m, 3h, ar) ; 7.19 (m, 2h, ar) ; 6.96 (m, 2h, ar) ; 5.79 (s, 2h, ch2), 3.97 (m, 1h, ch) ; 1.800.92 (m, 12h, cyclohexyl + ch3). c nmr (cdcl3) : 164.39, 162.75, 162.12, 150.90, 142.47, 140.56, 138.85, 134.66, 133.26, 128.86, 127.92, 127.63, 125.56, 124.55, 122.8, 116.33, 115.28, 55.78, 49.47, 44.88, 34.21, 32.20, 30.54, 22.56. h nmr (cdcl3) : 10.01 and 9.59 (2 m, 1h, nh) ; 8.91 (m, 2h, ar) ; 8.18 (s, 1h, ar) ; 7.56 (m, 3h, ar) ; 7.21 (m, 2h, ar) ; 6.97 (m, 2h, ar) ; 5.81 (s, 2h, ch2), 4.26 and 3.92 (2 m, 1h, ch) ; 1.820.92 (m, 12h, cyclohexyl + ch3). c nmr (cdcl3) : 164.82, 162.72, 162.14, 161.87, 150.70, 149.24, 142.55, 140.65, 139.67, 136.26, 132.55, 130.42, 129.16, 124.27, 116.88, 115.69, 115.29, 49.47, 46.16, 44.88, 34.21, 33.25, 32.20, 31.28, 30.54, 29.86, 22.56, 21.80. hrms - esi : m / z calcd for c29h27f2n3o2 [m+h ], 488.2150 ; found 488.2137. h nmr (cdcl3) : 9.99 and 9.60 (2 m, 1h, nh) ; 8.98 (m, 2h, ar) ; 8.29 (s, 1h, ar) ; 7.49 (m, 3h, ar) ; 6.99 (m, 2h, ar) ; 6.80 (d, j = 3.2 hz, 1h, ar) ; 6.56 (m, 1h, ar) ; 5.79 (s, 2h, ch2), 4.27 and 3.93 (2 m, 1h, ch) ; 1.830.91 (m, 12h, cyclohexyl + ch3). c nmr (cdcl3) : 164.39, 162.75, 162.12, 154.33, 150.90, 142.47, 142.09, 140.56, 134.66, 133.26, 128.86, 124.55, 122.8, 116.33, 115.28, 109.35, 107.89, 49.40, 46.26, 44.88, 34.31, 33.28, 32.00, 31.78, 30.24, 29.67, 22.50, 21.76. hrms - esi : m / z calcd for c27h26fn3o3 [m+h ], 460.2036 ; found 460.2023. compounds 22-trans and 22-cis were obtained from derivative 22 by flash chromatography on a silica gel (toluene / ethyl acetate 8:1). yield 33% ; ms 459 m / z (m) ; mp 175178 c. h nmr (cdcl3) : 9.60 (m, 1h, nh) ; 8.98 (m, 2h, ar) ; 8.29 (s, 1h, ar) ; 7.49 (m, 3h, ar) ; 6.99 (m, 2h, ar) ; 6.80 (d, j = 3.2 hz, 1h, ar) ; 6.56 (m, 1h, ar) ; 5.79 (s, 2h, ch2), 3.93 (m, 1h, ch) ; 1.830.91 (m, 12h, cyclohexyl + ch3). c nmr (cdcl3) : 164.39, 162.75, 162.12, 154.33, 150.90, 142.47, 142.09, 140.56, 134.66, 133.26, 128.86, 124.55, 122.8, 116.33, 115.28, 109.35, 107.89, 46.26, 44.88, 33.28, 31.78, 29.67, 21.76. yield 27% ; ms 459 m / z (m) ; mp 168170 c. h nmr (cdcl3) : 9.99 (m, 1h, nh) ; 8.98 (m, 2h, ar) ; 8.29 (m, 1h, ar) ; 7.49 (m, 3h, ar) ; 6.99 (m, 2h, ar) ; 6.80 (d, j = 3.2 hz, 1h, ar) ; 6.56 (m, 1h, ar) ; 5.79 (s, 2h, ch2), 4.27 (m, 1h, ch) ; 1.830.91 (m, 12h, cyclohexyl + ch3). c nmr (cdcl3) : 164.39, 162.75, 162.12, 154.33, 150.90, 142.47, 142.09, 140.56, 134.66, 133.26, 128.86, 124.55, 122.8, 116.33, 115.28, 109.35, 107.89, 49.40, 44.88, 34.31, 32.00, 30.24, 22.50. h nmr (cdcl3) : 10.04 and 9.63 (2 m, 1h, nh) ; 8.90 (m, 2h, ar) ; 8.18 (s, 1h, ar) ; 7.56 (m, 2h, ar) ; 7.06 (m, 2h, ar) ; 4.77 (t, j = 6.7 hz, 2h, ch2), 4.22 and 3.96 (2 m, 1h, ch) ; 3.89 (s, 3h, ch3) ; 3.68 (m, 4h, morpholine) ; 2.73 (m, 6h, morpholine + ch2) ; 1.950.87 (m, 12h, cyclohexyl + ch3). c nmr (cdcl3) : 162.45, 161.87, 161.06, 152.45, 148.89, 139.93, 128.72, 128.57, 125.98, 124.45, 116.31, 115.36, 114.81, 67.44, 56.34, 55. 91, 54.32, 49.63, 45.82, 39.60, 34.16, 33.12, 32.25, 31.34, 30.41, 29.90, 22.43, 21.78. hrms - esi : m / z calcd for c29h36n4o4 [m+h ], 505.2815 ; found 505.2801. h nmr (cdcl3) : 10.02 and 9.61 (2 m, 1h, nh) ; 8.94 (m, 2h, ar) ; 8.20 (s, 1h, ar) ; 7.41 (d, j = 4.4 hz, 1h, ar) ; 7.277.14 (m, 2h, ar) ; 4.80 (t, j = 6.6 hz, 2h, ch2), 4.28 and 3.97 (2 m, 1h, ch) ; 3.69 (m, 4h, morpholine) ; 2.74 (m, 6h, morpholine + ch2) ; 1.820.88 (m, 12h, cyclohexyl + ch3). c nmr (cdcl3) : 162.45, 161.87, 152.45, 148.89, 139.93, 138.31, 127.92, 127.65, 125.53, 122.68, 124.45, 116.31, 115.36, 67.44, 56.34, 54.32, 49.63, 45.82, 39.60, 34.16, 33.12, 32.25, 31.34, 30.41, 29.90, 22.63, 21.80. hrms - esi : m / z calcd for c26h32n4o3s [m+h ], 481.2273 ; found 481.2270. h nmr (cdcl3) : 10.00 and 9.62 (2 m, 1h, nh) ; 8.87 (m, 2h, ar) ; 8.17 (s, 1h, ar) ; 7.57 (m, 2h, ar) ; 7.20 (m, 2h, ar) ; 4.79 (t, j = 6.6 hz, 2h, ch2), 4.27 and 3.96 (2 m, 1h, ch) ; 3.68 (m, 4h, morpholine) ; 2.68 (m, 6h, morpholine + ch2) ; 1.800.89 (m, 12h, cyclohexyl + ch3). c nmr (cdcl3) : 164.55, 162.24, 161.68, 152.35, 148.92, 139.88, 132.21, 129.32, 125.98, 124.65, 116.31, 116.13, 115.30, 67.48, 56.65, 54.42, 49.33, 45.75, 39.45, 34.09, 33.22, 32.64, 31.54, 30.22, 29.89, 22.64, 21.80. hrms - esi : m / z calcd for c28h33fn4o3 [m+h ], 493.2615 ; found 493.2601. h nmr (cdcl3) : 10.04 and 9.64 (2 m, 1h, nh) ; 8.99 (m, 2h, ar) ; 8.28 (s, 1h, ar) ; 7.57 (d, j = 3.2 hz, 1h, ar) ; 6.80 (d, j = 3.2 hz, 1h, ar) ; 6.56 (m, 1h, ar) ; 4.77 (t, j = 6.7 hz, 2h, ch2), 4.22 and 3.96 (2 m, 1h, ch) ; 3.69 (m, 4h, morpholine) ; 2.77 (m, 6h, morpholine + ch2) ; 1.820.92 (m, 12h, cyclohexyl + ch3). c nmr (cdcl3) : 162.31, 162.00, 152.67, 148.920, 142.77, 139.91, 124.46, 122.87, 116.33, 115.31, 109.67, 107.54, 67.68, 56.33, 54.21, 49.21, 45.65, 39.59, 34.15, 33.18, 32.57, 31.32, 30.49, 29.99, 22.63, 21.82. hrms - esi : m / z calcd for c26h32n4o4 [m+h ], 465.2502 ; found 465.2490. the new compounds were evaluated in cb1r and cb2r binding assays using membranes from hek-293 cells transfected with cdnas encoding the human recombinant cb1r (bmax = 2.5 pmol / mg protein) and human recombinant cb2r (bmax = 4.7 pmol / mg protein) (perkin - elmer, italy). these membranes were incubated with [h]-()-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol ([h]cp-55,940) (0.14 nm / kd = 0.18 nm and 0.084 nm / kd = 0.31 nm for cb1r and cb2r, respectively) as high - affinity ligand and displaced with 100 nm (r)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (win-55,212 - 2) as heterologous competitor for nonspecific binding (ki = 9.2 and 2.1 nm, respectively, for cb1r and cb2r). cb1r binding protocol involves the use of the same solution buffer used for both incubation and washing reaction (tris - hcl, 50 mm ; edta, 2.5 mm ; mgcl2, 2.5 mm ; bsa, 0.5 mg / ml at ph 7.4), 0.4 nm for [h]cp-55,940, test compounds (concentrations from 0.001 to 10 m), and finally 8 g / sample membrane in a total volume of 200 l. binding assays were carried out with two different buffers : incubation buffer (tris - hcl, 50 mm ; egta, 2.5 mm ; mgcl2, 5 mm ; bsa, 1 mg / ml at ph 7.4) and washing buffer (tris - hcl, 50 mm ; egta, 2.5 mm ; mgcl2, 5 mm ; bsa, 2% at ph 7.4). the assay mixture contained incubation buffer, 0.4 nm [h]cp-55,940, test substances (concentrations from 0.001 to 10 m), and 4 g / sample membrane in a total assay volume of 600 l. assay tubes were prepared in duplicate and incubated for 90 min at 30 c. the reaction was terminated by addition of ice - cold buffer followed by rapid filtration under vacuum through whatman gf / c filters (pretreated for 2 h with 0.05% aqueous polyethyleneimine) using a 12-well harvester from millipore. after washing, radioactivity associated with the filters was counted on a liquid scintillation analyzer (tri - carb 2100 tr, perkin - elmer). specific binding was determined by subtracting nonspecific binding from total binding in the absence of competing ligand. the percentage displacement of specific binding was calculated for the amount of radiolabel bound in the presence of unlabeled displacing ligand. displacement ic50 values were determined by linear regression analysis of log concentration percent displacement data using graphpad prism. ki values were calculated by applying the cheng prusoff equation, ki = ic50/(1 + l / kd), where l is the concentration of the radioligand, ic50 is the concentration of drug causing 50% inhibition of specific radioligand binding, and kd is the dissociation constant of the radioligand receptor complex. u2os cells (osteosarcoma cell line) permanently expressing h - cb2r and arr2-gfp (green fluorescent protein) were obtained from drs. they were maintained at 37 c in a humidified atmosphere containing 5% co2 in dulbecco s modified eagle medium nutrient mixture f-12 ham, supplemented with 10% fetal bovine serum, 0.6% zeocin, and 400 mg / ml of g418. cells were then harvested using trypsin - edta (gibco catalog number 25300 - 054), and viable cells were assessed using trypan blue dye exclusion. u2os cells permanently expressing h - cb2r and arr2-gfp were detached using trypsin - edta, seeded onto glass coverslips at 8085% confluence, and placed in 24-well plates (bd falcon). after incubation at 37 c (5% co2, 95% relative humidity) overnight, cells were washed with hanks s balanced salt solution (hbss) before drug application. test compounds and reference cannabinoid compounds were dissolved in dmso, and dilutions were made in hbss. in order to detect agonist - stimulated redistribution of arr2-gfp, the cells were stimulated with various concentrations drug at room temperature for 40 min. then the suspension was removed, paraformaldehyde (4% in hbss, p / v) was added, and the incubation continued at room temperature for 25 min. finally the cells were washed with pbs three times and once with double - distilled water. the antagonism protocol included 15 min of pre - incubation with the antagonist, followed by a 40 min co - incubation of antagonist and agonist (30 nm win-55,212 - 2). glass coverslips were mounted onto slides and imaged using a fluorescence microscope (nikon e1000 ; tokyo, japan) using a 40 oil objective and 488 nm excitation for gfp. the redistribution of diffuse -arrestin - gfp from the cytoplasm to agonist- or antagonist - occupied receptor - containing pits or vesicles was imaged using a fluorescence microscope (nikon e1000 ; using a 40 oil objective and 488 nm excitation for gfp, tokyo, japan). the rgb color images captured from the fluorescent microscope were transformed into 8-bit gray - scale images using the automate - batch function in adobe photoshop cs5. to quantify arr2-gfp aggregates, gray - scale images were processed through imagej software (http://rsbweb.nih.gov/ij/), using a custom - written plug - in provided by pingwei zhao (temple university). response equation in graphpad prism version 5.0 (graphpad, san diego, ca). activity values were normalized to the agonist s response (30 nm win-55,212 - 2 was considered as 100%). these assays were performed using lance ultra camp kit (catalog number trf0262 ; perkin - elmer inc., u2os cells expressing the hcb2r were detached using trypsin - edta, washed with hbss, and counted, and cell viability was determined using trypan blue stain. cells were resuspended in stimulation buffer (hbss, 1x ; bsa stabilizer, 0.1% ; ibmx, 0.5 mm ; hepes, 5 mm ; ph 7.4) at a concentration of 600 cells/l. in order to detect the agonist - induced reduction in camp levels, 5 l of the cell suspension (3000 cells / well) was stimulated with forskolin (10 m final concentration) and with various concentrations of test ligands in white optiplate-384 wells at room temperature for 30 min. functional antagonism of the cannabinoid cb2r antagonist response was measured by incubating the suspension cells with drug dilutions, forskolin (10 m final concentration), and the reference agonist win-55,212 - 2 (30 nm final concentration) at room temperature for 30 min. after the incubation, 5 l of europium chelated labeled camp tracer solution in detection buffer and then 5 l of the camp - specific monoclonal antibodies (labeled with ulight - dye) solution in detection buffer were added to the wells. the reaction was allowed to incubate for 1 h at room temperature in the dark. time - resolved fluorescence signals were detected on an envision multiplate reader (perkin - elmer, ca, usa) at 615 and 665 nm emission. the amounts of camp produced in the stimulated cells were determined according to the camp standard curves. antagonism in the camp assay has been expressed as percent of inhibition of win-55,212 - 2. inhibition curves were analyzed by nonlinear regression using graphpad prism version 5.0 software (graphpad, san diego, ca), and data were fitted to sigmoidal concentration response curves to obtain ic50 values., the logarithmic value of agonist concentrations is plotted against the tr - fret signal normalized to the response of forskolin. here, the ballesteros weinstein numbering system for gpcr amino acid rersidues is used. in this numbering system, the label 0.50 is assigned to the most highly conserved class a residue in each transmembrane helix (tmh). this is preceded by the tmh number. in this system, for example, the most highly conserved residue in tmh6 is p6.50. the residue immediately before this would be labeled 6.49, and the residue immediately after this would be labeled 6.51. when referring to a specific cb2 residue, the ballesteros weinstein name is followed by the absolute sequence number given in parentheses (e.g., k3.28(109)) ; however, when referring to a highly conserved residue among class a gpcrs (and not a specific residue in cb2), only the ballesteros initial conformational analyses of these compounds were performed using the semiempirical method am1 encoded in spartan08. conformational searches were performed (using 38-fold rotations) for each rotatable bond. fock calculations at the 6 - 31 g level, except for 17 and 18, which required 6 - 311 g to accommodate a bromine atom. to calculate the difference in energy between the global minimum energy conformer of each compound and its final docked conformation, rotatable bonds in the global minimum energy conformer were driven to their corresponding value in the final docked conformation, and the single - point energy of the resultant structure was calculated at the hf 6 - 31 g level, or for 17 and 18 at the 6 - 311 g level. complete details on the generation of the inactive and activated state cb2r models used here are available in our previous publication. the crystal structure of the class a gpcr, rhodopsin in the dark state was used as the template for the creation of our cb2r inactive state model. this template was chosen because no mutations or modifications were made to its structure for crystallization. a trp forms an aromatic stacking interaction with y5.39, influencing the ec positions of tmh3 - 4 - 5. the initial homology model was refined by calculating the low free energy conformations for any tmh with an important sequence divergence from rhodopsin and replacing the corresponding helix from the initial model with one that more accurately reflects the sequence dictated tmh geometries in cb2r. this includes tmh2 (gg helix distorting motif in rho vs no pro or gg in cb2r) and tmh5 (pro at 5.50 in rho vs no pro at 5.50 in cb2r). the resultant cb2r model has been tested using results from substituted cysteine accessibility studies to identify binding pocket facing residues, from mutation studies of key ligand interactions sites, and from covalent labeling studies of cb2r that support a lipid entry pathway for cb2r ligands. to permit adjustment to a lipid bilayer environment, the resultant model was pre - equilibrated in a stearoyl - docosahexaenoylphosphatidylcholine (sdpc) bilayer for 300 ns. while the toggle switch residue, w6.48(258), remained in its inactive state g dihedral angle after the equilibration in sdpc, some notable changes did occur during this equilibration. the r3.50(131) and d6.30(240) salt bridge at the intracellular ends of tmhs3/6 (analogous to the r3.50(135)/e6.30(247) salt bridge in the dark state of rhodopsin) rearranged quickly to form a salt bridge between r3.55(136) and d6.30(240), with y3.51(132) supporting the salt bridge by hydrogen bonding to the exposed backbone carbonyl of l6.29(239). a second notable change was the development of additional helical turns in the ic-3 (tmh5-tmh6) loop after the original end of tmh5. the cb2r activated state model (r) used here for docking studies was produced from the inactive state model described above via a multimicrosecond - long molecular dynamics simulation of the interaction of the endogenous cb2r ligand, 2-ag, with cb2r in a palmitoyl - oleoyl - phosphatidylcholine (popc) bilayer. in these simulations, 2-ag entered the binding pocket via the lipid bilayer between tmh6 and tmh7 and activated the cb2r. ligand entry resulted in changes on the intracellular end of the receptor. here the r3.55(136)/d6.30(240) ionic lock was broken as tmh6 straightened and moved its ic end away from the tmh bundle. ligand entry also resulted in changes in the binding pocket, as toggle switch residue w6.48(258) underwent a torsion angle change from g to trans. this change was transitory, with w6.48(258) reverting to a g+. the r model used for docking studies here was taken from the section of the trajectory in which the w6.48(258) was trans.(31) the inactive state model was used to dock compounds 17, 18, and 23, while a1, a2, 5, and 14 were docked in the activated state model described above. in addition, to probe the origins of the antagonism vs agonism of 23 and a1, each compound was docked in our cb2r inactive state model., portland, or) was used to explore possible binding conformations or receptor site interactions with flexible docking. because s7.39(285) has been shown to be a ligand interaction site in cb2r, s7.39(285) was defined as a required interaction during the initial flexible docking procedure. glide was used to generate a grid based on the centroid of the ligand in the binding site. any hydrophobic region defined in the grid generation that contacted the ligand was selected as important to the flexible docking procedure. the box for flexible docking was defined to be 26 in the x, y, and z dimensions. extra precision (xp) was selected with scaling of vdw radii and flexible docking invoked. a second glide docking study was initiated in which k3.28(109) and s7.39(285) were defined as required interactions during the flexible docking procedure. this second run resulted in improved glide scores, particularly for ligands with higher cb2r binding affinities. for each receptor ligand complex, the complex with the best glide score was minimized using the opls2005 all - atom force field in macromodel 9.9 (schrodinger inc.). an 8.0 nonbonded cutoff (updated every 10 steps), a 20.0 electrostatic cutoff, and a 4.0 hydrogen bond cutoff were used in each stage of the calculation. the first stage consisted of 3500 steps of polak ribier conjugate gradient minimization using a distance - dependent dielectric function with a base constant of 2. no harmonic constraints were placed on the side chains, but 100 kj / mol torsional constraints were applied to hold all the backbone / torsion angles. during the second stage of 500 steps, all torsional constraints were released. to relax the loops, an additional 1000-step polak ribier conjugate gradient minimization of the loop regions was performed. the loop and termini regions were left free, while the transmembrane regions were not allowed to move during this final minimization. an 8.0 extended nonbonded cutoff (updated every 10 steps), 20.0 electrostatic cutoff, and 4.0 hydrogen bond cutoff were used in this calculation, and the generalized born / surface area (gb / sa) continuum solvation model for water available in macromodel was employed. the structures of ligands were built in spartan08 (wave function, inc., initial conformational analyses of these compounds were performed using the semiempirical method am1 encoded in spartan08. conformational searches were performed (using 38-fold rotations) for each rotatable bond. fock calculations at the 6 - 31 g level, except for 17 and 18, which required 6 - 311 g to accommodate a bromine atom. to calculate the difference in energy between the global minimum energy conformer of each compound and its final docked conformation, rotatable bonds in the global minimum energy conformer were driven to their corresponding value in the final docked conformation, and the single - point energy of the resultant structure was calculated at the hf 6 - 31 g level, or for 17 and 18 at the 6 - 311 g level. complete details on the generation of the inactive and activated state cb2r models used here are available in our previous publication. the crystal structure of the class a gpcr, rhodopsin in the dark state was used as the template for the creation of our cb2r inactive state model. this template was chosen because no mutations or modifications were made to its structure for crystallization. a trp forms an aromatic stacking interaction with y5.39, influencing the ec positions of tmh3 - 4 - 5. the initial homology model was refined by calculating the low free energy conformations for any tmh with an important sequence divergence from rhodopsin and replacing the corresponding helix from the initial model with one that more accurately reflects the sequence dictated tmh geometries in cb2r. this includes tmh2 (gg helix distorting motif in rho vs no pro or gg in cb2r) and tmh5 (pro at 5.50 in rho vs no pro at 5.50 in cb2r). the resultant cb2r model has been tested using results from substituted cysteine accessibility studies to identify binding pocket facing residues, from mutation studies of key ligand interactions sites, and from covalent labeling studies of cb2r that support a lipid entry pathway for cb2r ligands. to permit adjustment to a lipid bilayer environment, the resultant model was pre - equilibrated in a stearoyl - docosahexaenoylphosphatidylcholine (sdpc) bilayer for 300 ns. while the toggle switch residue, w6.48(258), remained in its inactive state g dihedral angle after the equilibration in sdpc, some notable changes did occur during this equilibration. the r3.50(131) and d6.30(240) salt bridge at the intracellular ends of tmhs3/6 (analogous to the r3.50(135)/e6.30(247) salt bridge in the dark state of rhodopsin) rearranged quickly to form a salt bridge between r3.55(136) and d6.30(240), with y3.51(132) supporting the salt bridge by hydrogen bonding to the exposed backbone carbonyl of l6.29(239). a second notable change was the development of additional helical turns in the ic-3 (tmh5-tmh6) loop after the original end of tmh5. the cb2r activated state model (r) used here for docking studies was produced from the inactive state model described above via a multimicrosecond - long molecular dynamics simulation of the interaction of the endogenous cb2r ligand, 2-ag, with cb2r in a palmitoyl - oleoyl - phosphatidylcholine (popc) bilayer. in these simulations, 2-ag entered the binding pocket via the lipid bilayer between tmh6 and tmh7 and activated the cb2r. ligand entry resulted in changes on the intracellular end of the receptor. here the r3.55(136)/d6.30(240) ionic lock was broken as tmh6 straightened and moved its ic end away from the tmh bundle. ligand entry also resulted in changes in the binding pocket, as toggle switch residue w6.48(258) underwent a torsion angle change from g to trans. this change was transitory, with w6.48(258) reverting to a g+. the r model used for docking studies here was taken from the section of the trajectory in which the w6.48(258) was trans.(31) the inactive state model was used to dock compounds 17, 18, and 23, while a1, a2, 5, and 14 were docked in the activated state model described above. in addition, to probe the origins of the antagonism vs agonism of 23 and a1, each compound was docked in our cb2r inactive state model., portland, or) was used to explore possible binding conformations or receptor site interactions with flexible docking. because s7.39(285) has been shown to be a ligand interaction site in cb2r, s7.39(285) was defined as a required interaction during the initial flexible docking procedure. glide was used to generate a grid based on the centroid of the ligand in the binding site. any hydrophobic region defined in the grid generation that contacted the ligand was selected as important to the flexible docking procedure. the box for flexible docking was defined to be 26 in the x, y, and z dimensions. extra precision (xp) was selected with scaling of vdw radii and flexible docking invoked. a second glide docking study was initiated in which k3.28(109) and s7.39(285) were defined as required interactions during the flexible docking procedure. this second run resulted in improved glide scores, particularly for ligands with higher cb2r binding affinities. for each receptor ligand complex, the complex with the best glide score was minimized using the opls2005 all - atom force field in macromodel 9.9 (schrodinger inc.). an 8.0 nonbonded cutoff (updated every 10 steps), a 20.0 electrostatic cutoff, and a 4.0 hydrogen bond cutoff were used in each stage of the calculation. the first stage consisted of 3500 steps of polak ribier conjugate gradient minimization using a distance - dependent dielectric function with a base constant of 2. no harmonic constraints were placed on the side chains, but 100 kj / mol torsional constraints were applied to hold all the backbone / torsion angles. during the second stage of 500 steps, all torsional constraints were released. to relax the loops, an additional 1000-step polak ribier conjugate gradient minimization of the loop regions was performed. the loop and termini regions were left free, while the transmembrane regions were not allowed to move during this final minimization. an 8.0 extended nonbonded cutoff (updated every 10 steps), 20.0 electrostatic cutoff, and 4.0 hydrogen bond cutoff were used in this calculation, and the generalized born / surface area (gb / sa) continuum solvation model for water available in macromodel was employed. | we have recently identified 1,8-naphthyridin-2(1h)-one-3-carboxamide as a new scaffold very suitable for the development of new cb2 receptor potent and selective ligands. in this paper we describe a number of additional derivatives in which the same central scaffold has been variously functionalized in position 1 or 6. all new compounds showed high selectivity and affinity in the nanomolar range for the cb2 receptor. furthermore, we found that their functional activity is controlled by the presence of the substituents at position c-6 of the naphthyridine scaffold. in fact, the introduction of substituents in this position determined a functionality switch from agonist to antagonists / inverse agonists. finally, docking studies showed that the difference between the pharmacology of these ligands may be in the ability / inability to block the toggle switch w6.48(258) (1 g+ trans) transition. |
in biomedical scientific investigations, expositions of findings are conceptually simplest when they comprise comparisons of discrete groups of individuals or involve discrete features or characteristics of individuals. but the descriptive benefits of categorization become outweighed by their limitations in studies involving dose - response relationships, as in many teratogenic and environmental exposure studies. this article addresses a pair of categorization issues concerning the effects of prenatal alcohol exposure that have important public health consequences : the labeling of individuals as fetal alcohol syndrome (fas) versus fetal alcohol effects (fae) or alcohol - related neurodevelopmental disorder (arnd), and the categorization of prenatal exposure dose by thresholds. we present data showing that patients with fas and others with fae do not have meaningfully different behavioral performance, standardized scores of iq, arithmetic and adaptive behavior, or secondary disabilities. similarly overlapping distributions on measures of executive functioning offer a basis for identifying alcohol - affected individuals in a manner that does not simply reflect iq deficits. at the other end of the teratological continuum, we turn to the reporting of threshold effects in dose - response relationships. here we illustrate the importance of multivariate analyses using data from the seattle, washington, longitudinal prospective study on alcohol and pregnancy. relationships between many neurobehavioral outcomes and measures of prenatal alcohol exposure are monotone without threshold down to the lowest nonzero levels of exposure, a finding consistent with reports from animal studies. in sum, alcohol effects on the developing human brain appear to be a continuum without threshold when dose and behavioral effects are quantified appropriately.imagesfigure 1figure 3 |
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systemic lupus erythematosus (sle) is a systemic autoimmune disease that is known to be associated with polyclonal b cell hyperreactivity. a combination of genetic factors, environmental influences, and sex hormones may contribute to the susceptibility to the disease. while t cells have been considered to play a central role in the pathogenesis of sle before, emerging evidences indicate that b cells may be a more important player than t cells in the pathogenesis of sle [1, 35 ]. lipid rafts (lrs), the specialized cholesterol- and glycosphingolipid - rich microdomains in the cellular membrane, have been reported to play an important role in the activation of b cells, including effects on b cell antigen receptor- (bcr-) initiated signal transduction, endocytosis of bcr - antigen complexes, loading of antigenic peptides onto mhc class ii molecules, and receipt of helper signals via the cd40 receptor [6, 7 ]. lipid rafts are also linked to the actin cytoskeleton which is important for lymphocyte antigen presenting. actin is a multifunctional protein that forms microfilaments presented either as a free monomer called g - actin or as a polymer microfilament called f - actin. via polymerization and depolymerization dynamic regulation of f - actin cytoskeleton is critical to numerous physical cellular processes, including cell motility, cell division and cytokinesis, cell signaling, and the establishment and maintenance of cell junctions and cell shape. many of these processes are mediated by extensive and intimate interactions of actin with cellular membranes. the actin cytoskeleton has also been reported to be involved in b cell activation by participating in the processing of the maturation of mhc class ii molecules, internalization of bcr, and interaction between fcri and lipid raft components [8, 9 ]. abnormal expression of lipid rafts and f - actin on t cells in sle patients has been studied well [1013 ]. the expression pattern of lrs and f - actin on b cells in sle patients remains not quite clear. leflunomide (lef) is one of the most important immunosuppressive drugs and has been extensively used in the therapy of rheumatoid arthritis in recent years. it also showed great success in treatment of sle in both animal models and patients, though it was still not recommended as one choice of therapeutic drugs for sle until now. following intestinal absorption, lef is rapidly converted into its active metabolite a771726 that has been known to inhibit dihydroorotate - dehydrogenase (dhodh) and tyrosine kinases, leading to decreased proliferation of t and b cells. however, the specific mechanism of how lef regulates lymphocyte activation remained unclear. whether lef is involved in modulation of lrs and f - actin expression to exert its therapeutic effect on sle is not fully understood. a better understanding about the role of lef in sle will help us in using it appropriately. to clarify the changes in b cell subsets and lrs and f - action expression pattern on b cells and determine the possible role of lef in these changes in the patients with sle, we measured cd19, cd38, cd95, cd27, igd, gm1, and f - actin expression levels and their patterns on b cells from sle patients. our results suggest that there were abnormalities at b cell subsets and expression of lrs and f - actin on b cells, which could be modified by a771726 in sle. 54 sle patients (51 females, 3 males ; average age : 32.06 11.38), 15 sex - matched (all females) primary sjgren 's syndrome (pss) patients, and 20 age- and sex - matched healthy volunteers (19 females, 1 male ; average age : 30.60 10.78) who served as controls were enrolled for this study. the diagnosis of sle was according to the 1997 revised american college of rheumatology (acr) criteria for the classification of sle. the diagnosis of pss was according to the 2002 american - european consensus group criteria. disease activity in the sle patients was assessed using the sle disease activity index (sledai). in our cohort, sle patients were divided into two subgroups based on sledai including 37 active sle (sledai > 6) and 17 inactive sle (sledai 5). all patients either were not exposed to or have discontinued for at least 2 weeks glucocorticoid and immunosuppressant drugs before recruitment into this study. this study was performed according to the principles of the declaration of helsinki and each participant completed written informed consent before measurements. this study was also approved by the local ethics committee (institutional review board of guangdong general hospital). 15 ml edta - treated blood samples were obtained after an overnight fasting. peripheral blood mononuclear cells (pbmcs) were isolated by density gradient centrifugation over ficoll - paque plus. for immunofluorescence staining, the following monoclonal antibodies (mabs) were used : cd5-pe (bd pharmingen), cd19-fitc / cy5 (ebioscience), cd27-pe (ebioscience), cd38-cy7 (biolegend), cd95-pe (ebioscience), and igd - fitc (invitrogen). to define b cell subpopulations, four - color immunofluorescence analysis was performed on the same day by flow cytometry (bechman coulter inc.). the fetched cells suspension was gated for lymphocyte and at least 10,000 lymphocyte events were acquired per sample. b cells were isolated from pbmcs by positive selection using macs anti - cd19 microbeads and ms columns (miltenyi biotec, gmbh, germany) following the manufacturer 's instructions. flow cytometry consistently showed that the percentage of b cells in the isolated subpopulation was > 90% as determined by anti - cd19 mab staining. b lymphocytes were cultured in serum - free rpmi-1640 medium at 37c for 4 hours, with or without a771726 (kindly provided by cinkate corporation, china) (at 0 g / ml, 15 g / ml, 30 g / ml, 45 g / ml, and 60 g / ml). the binding of cholera toxin b subunit (ctb) (sigma) is frequently used to identify lipid rafts, and the capacity for binding ctb is upregulated during b cell activation. to analyze lipid raft, we stained cells with fluorescence - labeled ctb, which binds to the raft - associated glycosphingolipid gm1, previously shown to be a reliable marker for the detection of lipid raft domains. in vitro a771726-treated b cells were fixed with 2% paraformaldehyde (pfa) at room temperature (rt) for 15 min. fixed cells were resuspended in 100 l phosphate buffered saline (pbs) and then stained with ctb - fitc (final concentration 2 g / ml). at least 5000 cells were analyzed. to analyze surface cd38 or cd95 coexpression with lipid rafts in b cells, pbmcs were isolated and were stained with fluorochrome - conjugated mouse monoclonal anti - cd19, anti - cd38, and anti - cd95. cells were fixed with 2% pfa at rt for 15 min and subsequently stained with ctb - alexa flour 488 (molecular probes inc.) (final concentration : 10 g / ml). ex vivo and in vitro a771726-treated b cells were washed by pbs and added to poly - l - lysine cover slips and mounted to slides. cells were fixed with 2% pfa at rt for 15 min. staining for f - actin membrane staining for lipid rafts was achieved with ctb - fitc (final concentration : 10 g / ml) at 4c for 30 min. the colocalization of lipid rafts and f - actin was quantified using the colocalization tool in the leica software. differences between two groups were compared using the nonparametric mann - whiney test for unpaired data and wilcoxon test for paired data. initially, an analysis of cd5 expression in peripheral b cells was carried out in 20 active sle patients and 11 healthy subjects. the result showed that the percentage of b cells in lymphocytes was significantly increased in sle patients compared with normal subjects (median 16.7% (range : 2.548.0) versus 10.6% (range : 5.717.2)) (p = 0.01). but there was no difference in the frequency of cd5 expression on b cells between sle patients (median 24.9% (range : 6.968.3)) and normal individuals (median 27.1% (range : 10.856.7)) (p = 0.409). further analysis of the expression of surface igd and cd27 in b cell subsets in 17 active sle patients and 10 healthy subjects showed no difference in cd27 b cells and igd b cells between sle and normal control (cd27 b cells : median 45.1% (range : 10.980.5) versus median 38.9% (range : 27.570.1), p = 0.941 ; igd b cells : median 52.8% (range : 15.688.8) versus median 64.1% (range : 47.772.4), p = 0.711). however, a decrease in cd27igd b cell subset was observed in patients with sle (median 7.7% (range : 2.032.6) versus 15.1% (range : 9.132.6)) (p = 0.031). the proportion of cd27igd b cell in patients with lupus nephritis (n = 10, median 5.0% (range : 2.011.1)) was lower than those in sle patients without renal involvement (n = 7, median 11.9% (range : 4.732.6)) (p = 0.032) (figure 1(a)). but there was no difference in the percentage of cd27igd b cells between sle patients and normal control individuals. finally, we analyzed cd38 and cd95 expression in b cells surface. there was no difference in the percentage of cd95 b cells between sle patients and normal controls, but an increased expression of cd38 b cells was found and the percentage of cd38cd95 b cells was significantly increased (figure 1(b)) in active sle patients. the lrs expression level in b cells was remarkably increased in sle patients (median 57.0% (range : 29.787.4), n = 38) compared with those in healthy controls (median 34.8% (range : 8.450.0), n = 11) (p < 0.001). the lrs level on b cells was significantly higher in active sle patients than in inactive sle patients (median 59.6% (range : 35.387.4), n = 18, versus median 50.7% (range : 29.762.7), n = 20) (p < 0.001) (figure 2(a)). in addition, the lrs level in b cells in inactive sle patients was still higher than that in healthy controls (p = 0.003). there was a positive correlation between the lrs level in b cells and sledai (r = 0.568, p < 0.001) (figure 2(b)). likewise, the lrs level in b cells showed a significant positive correlation with anti - dsdna antibody titer (r = 0.394, p = 0.028). no correlation was seen between the lrs level in b cells and blood igg level (r = 0.203, p = 0.291) or complement factor c3 level (r = 0.036, p = 0.843). analysis of cd38 and cd95 coexpression with lrs in pbmcs from 12 active sle patients and 11 healthy controls revealed a significant increased lrs expression on cd38 b cells in sle patients compared with healthy controls (figure 1(b)). interestingly, in active sle patients, lrs expression in cd38 b cells (median 36.1% (range : 15.147.3)) was significantly higher than that in cd38 b cells (median 10.5% (range : 4.131.7)), p < 0.001. the increased percentage of lrs expression in cd38 b cell was correlated with complement factor c3 level (r = 0.718, p = 0.013). to analyze the colocalization of lipid rafts and f - actin, purified b cells from 7 active sle patients, 6 pss patients, and 4 healthy controls confocal microscopy images revealed that, in b cells from healthy controls and pss, lrs were small and uniformly distributed on the plasma membrane. f - actin was found mainly in a cortical distribution around the perimeter of the cell. this pattern was different from the pattern seen in b cells from sle patients, which presented with stronger staining and an irregular large clustering of lrs companying with a decrease in f - actin level (figure 2(c)). our preliminary experiments found that a771726 did not obviously affect lrs level at the concentration of 15 or 30 g / ml. to investigate the effects of a771726 on lrs and f - actin on b cells, purified b cells from 10 active sle patients were cultured with or without a771726 (45 g / ml or 60 g / ml) at 37c for 4 hours. the percentage of ctb - binding lrs was significantly decreased after a771726 treatment and does not show significant difference between the concentration of 45 g / ml and the concentration of 60 g / ml (figure 3(a)). coexpression analysis of lrs and f - actin on sle b cells by confocal microscopy showed that treatment of a771726 reduced the cluster of lrs associated with reversed distribution of f - actin (figure 3(b)). several studies have documented the specific perturbations of b cell maturation and differentiation in sle and that alteration of b cell subsets and b cell phenotypes could play critical roles in sle diseases. [1, 22 ], but the possible inherent b cell abnormalities in sle patients remain to be defined clearly. our results showed that there was an increased frequency of cd38 b cells and decreased percentage of cd27igd b cells in sle patients, but no difference of cd5 b cells, cd27 b cells, and igd b cells between active sle patients and normal controls was found. moreover, sle patients with lupus nephritis had lower level of cd27igd b cells than sle patients without renal involvement. previous studies had described a new population of memory b cells lacking expression of both cd27 and igd, and increased frequency of this subset of b cells was associated with higher disease activity index. in our study, we observed no significant difference in the percentage of cd27igd b cells between sle patients and normal controls. however, our study showed that the percentages of cd38 b cells and cd38cd95 b cells were significantly increased in active sle patients, although there was no difference in the percentage of cd95 b cells between sle patients and normal controls. these different results may be attributed to sle hyperheterogeneity, ethnic 's heterogeneity, and grouping criteria heterogeneity. it is known that the increased expression of cd38 b cells is closely associated with memory b cells hyperactivation and differentiation, while the increased expression of cd95 on the b cells may induce b cell apoptosis. further study is required to reveal the clinical relevance of the increased cd38cd95 b cells in the pathogenesis and progression of sle. increased lipid raft expression on the plasma membrane of b cells may constitute a way to promote b cell activation. in our study, we found that the lipid raft levels in b cells were dramatically increased in sle patients, and the lipid raft levels in b cells were positively correlated with sledai and dsdna antibody titers. this suggests that the changes of lipid raft expression pattern on the b cells may contribute to b cell overactivation and disease activity in the sle patients. moreover, our study showed that the percentage of cd38gm1 b cells in sle was significantly increased. cd38 is associated with the synthetic metabolism of b cells and also participates in b cell activation and proliferation through regulating gene transcription and protein phosphorylation. cd38 expression can also lead to increase of intracellular ca level and enhanced bcr signaling. we speculated that the increased cd38 expression on b cells in sle patients might decrease the threshold of b cell activation by promoting b cell lipid raft overexpression. the distinct expansion of the cd38gm1 b cells in sle patients may be one of the major features of b cell inherent abnormality and related to sle pathogenesis. further mechanistic studies are required to reveal the relationship between cd38 and lipid rafts in b cells. depolymerization of the actin cytoskeleton could alter lipid rafts clustering and influence the threshold for cellular activation. whether lipid rafts and f - actin expression could be changed in b cells in sle has not been reported so far. we found that lipid rafts were irregularly distributed on the plasma membrane and showed aggregation in sle b cells, accompanied by a decrease in f - actin expression and abnormal distribution of f - actin, suggesting the abnormal expression of lrs and f - actin may contribute to the hyperactivation of b cells in sle patients. lef is a potent effective disease - modifying antirheumatic drug and has been recently demonstrated to be safe and effective for the treatment of sle and even refractory lupus and lupus nephritis [29, 30 ]. but it was still not recommended by the latest acr / eular treatment guidelines for sle. our data showed that lef treatment can reduce the lipid rafts levels and reverse the abnormal distribution of lipid rafts and f - actin of b cells in the active sle patients. this finding suggests that regulating the abnormal expression of lipid rafts and f - actin in b cells may be a new mechanism for lef to effectively treat sle and that modulation of abnormalities of lipid rafts and f - actin expression on b cells could be an important new target in the sle therapy. although this study was somewhat limited in statistical power because of the small sample size, our data showed that there were significant abnormalities in the b cell subsets and their expression pattern of lipid rafts and f - actin from sle patients. modifying these abnormalities may represent a new mechanism for lef in effective treatment of sle. the altered lipid rafts and f - actin expression pattern or their signaling pathways may be used as new targets in sle treatment. | purposes. to investigate the possible changes in b cell subsets and in b cell expression patterns of lipid rafts (lrs) and f - actin in patients with sle and whether leflunomide treatment may have effect on these changes. methods. the b cell subsets and lrs expression were determined by flow cytometry and confocal microscopy, and f - actin expression was examined by confocal microscopy. results. cd27+igd+ b cell subsets were significantly decreased while cd38+cd95 + b cell subsets increased in sle patients. the lrs levels of b cells were remarkably increased and positively correlated with sledai and anti - dsdna titer in sle patients. the expression level of lrs was significantly higher in cd38 + b cells than cd38 b cells and negatively correlated with c3 levels. the increased expression of lrs was associated with reduced expression of f - actin in the b cells from active sle patients. furthermore, in vitro treatment of the cells with a771726 reduced the expression level of lrs, attenuated the overaggregation of lrs, and normalized the distribution of f - actin. conclusions. there were abnormalities in b cell subsets and lrs and f - actin expression of b cell from sle patients. modulation of b cell expression of lrs and f - actin by lef could be a potential therapeutic target for sle. |
liposomes are a very attractive drug delivery system because they are physically and chemically well - characterized structures that can be delivered through almost all routes of administration and are biocompatible [13 ]. we have developed a submicron - sized (100200 nm) mucoadhesive liposomal system by modifying the liposome surface with a mucoadhesive polymer such as chitosan to achieve an oral peptide formulation [4, 5 ]. the effectiveness of polymer - modified liposomes was confirmed by the enhanced and prolonged pharmacological effect of peptide drugs such as insulin, which was orally administered in a polymer - coated liposomal form to rats. therefore, it is important to characterize the mucoadhesive properties of oral liposomal systems in vivo based on liposomal behavior in the body. in a previous study, we examined the mucosal layer of the rat intestine to detect organic dye - labeled liposomes by confocal laser scanning microscopy (clsm) after administering these particulate systems. in in vivo experiments, near - infrared (nir) optical imaging is a powerful tool for real - time observation of the dynamic behavior of liposomes because it is a minimally invasive, nonionizing method that permits sensitive deep tissue imaging [7, 8 ]. however, traditional nir dyes have several disadvantages for use as fluorescent probes, such as low solubility in aqueous solution, low quantum yield, and low photostability. semiconductor nanocrystals known as quantum dots (qds) are fluorescent nanoparticles with diameters of 110 nm. qds have been extensively investigated as optical probes for various biomedical applications in vitro and in vivo [1113 ]. in this present study, we prepared qd - loaded liposomes to examine liposomal behavior in the body after oral administration. therefore, the qds could be entrapped in the lipid bilayer and not in the inner aqueous phase. we also attempted to encapsulate hydrophobic qds with glutathione (gsh) surface modifications in the inner water layers of the liposome. the cellular uptake and cytotoxicity of these qd - loaded liposomes were evaluated using caco-2 cells. the feasibility of detecting liposomal qds in the gastrointestinal tract was investigated after oral administration to rats in an in vivo experiment. cdse / cdzns qds and gsh - modified cdse / cdzns qds (gsh - qds) were prepared and characterized as described previously [14, 15 ]. qd - loaded liposomes were prepared using a thin film hydration method. typically, a mixture of l--distearoylphosphatidylcholine (dspc ; nippon oil and fats, japan), stearylamine (sa, tokyo kasei, japan), dicetyl phosphate (dcp ; sigma, st. louis, mo, usa), and cholesterol (chol, sigma) were dissolved in chloroform in different molar ratios. the solvent was evaporated to dryness, and the lipid film was further dried under vacuum overnight. multilamellar vesicles were obtained by hydrating the lipid film using phosphate buffer (66.67 mm, ph 7.4). small unilamellar vesicles were obtained by probe sonication for 5 min in an ice water bath (sonifier 250 ; branson, mo, usa). the hydrophobic qds were loaded during the preparation of the thin lipid film, and the liposomes were prepared as aforementioned. for loading, unloaded qds and gsh - qds were removed by ultracentrifugation in a sucrose density gradient. the resolved fluorescent bands observed under uv illumination (las-3000 uv mini ; fuji film, tokyo, japan) were carefully recovered by manual pipetting and analyzed for qd encapsulation. the particle sizes were determined by dynamic light scattering (dls ; zetasizer nano zs, malvern, worcestershire, uk). the zeta potential of liposomes was measured by the laser doppler method (zetasizer nano zs). fluorescence spectra were recorded at room temperature using a fluorescence spectrophotometer (model fp-6600 ; jasco, japan). qd - loaded liposomes were observed by transmission electron microscopy (tem, jem-1200ex ; jeol, tokyo, japan). 1.5 german coverglass system (nalge nunc international, naperville, il, usa) at a density of 6.3 10 cells / cm. seven days after seeding, the growth medium was replaced with liposomal suspension, and cells were incubated for 1 h at 37c. cellular uptake was terminated by washing three times with ice - cold pbs and the cell monolayers were fixed with 0.5 ml of 4% paraformaldehyde. the fixed cells were observed using an lsm 700 clsm (carl zeiss, gottingen, germany). caco-2 cells were seeded in 96-well plates at a density of 3.15 10 cells / cm. the solution reagent contained mts and an electron - coupling reagent (phenazine ethosulfate). the cultures were further incubated for 2 h, and then 20 l aqueous one solution reagent was directly added to the culture wells. after 2 h of incubation, the absorbance of the produced color was measured with a microplate reader (mtb 120, corona electric, japan) at a wavelength of 490 nm. the quantity of the formazan product, as measured by the absorbance at 490 nm, was directly proportional to the number of living cells in the culture. male wistar rats (10 - 11 weeks old, slc, japan) were used in all in vivo studies. all experiments were approved and monitored by the institutional animal care and use committee of gifu pharmaceutical university, and experiments were performed in line with japanese legislation on animal studies. before the experiments, the rats were fasted for 24 h with free access to water. the mucosal association and penetration of qd - loaded liposomes were visualized by clsm using a method described previously. the freshly excised tissues were fixed in tissue - tek compound by immersion into liquid nitrogen. the molded samples were sectioned (10 m) using a cryomicrotome (leica cm, germany) and imaged by clsm. the average particle size of cdse / cdzns qds with a core - shell structure was approximately 6.8 nm as measured by dls. the fluorescence spectrum of cdse / cdzns qds in chloroform was measured at an excitation wavelength of 480 nm. emission was observed at 600 nm, and the spectrum had a symmetrical shape. in general, therefore, we attempted to embed hydrophobic (unmodified) qds into the liposomal lipid bilayer and examine the effects of qd concentration on liposomal properties. the particle size of qd - loaded liposomes was approximately 100 nm irrespective of the qd concentration (0400 nm). these liposomes exhibited a positive zeta potential (approximately 20 mv), derived from the sa amino acid group. to confirm that qd fluorescence is preserved after incorporation into the lipid bilayer and that the liposomal bilayers were fluorescent, the liposomal preparations with different qd concentrations were placed under a uv lamp immediately after formation (figures 1(a)1(e)). the liposomes lacking qds (negative control) emitted almost no fluorescence irrespective of ultracentrifugation (figures 1(a) and 1(b)). furthermore, the signals became stronger as the qd concentration increased (figures 1(c)1(e)). no difference was observed in the pattern of the fluorescence spectrum of qds after incorporation into the liposome compared with that of free qds (figure 1(g)), which suggested that qd degradation was not induced by the preparation process, and intact qds were embedded in the lipid bilayers. to visualize how qds are distributed inside the liposomal lipid bilayer, we collected and analyzed the images captured by tem (figure 1(f)). some of the liposomes were still qd - free, but some qds were loaded into the lipid bilayer. gsh is a tripeptide (-l - glutamyl - l - cysteinylglycine) and a biocompatible material that exists in most organs. gsh - qds were prepared by a method reported previously. in brief, the hydrophobic cdse / cdzns qds surrounding trioctylphosphine oxide and hexadecylamine molecules are dispersed in tetrahydrofuran (thf). then, a gsh solution is added to the qd solution in thf, and ligand exchange is performed at 60c. after the deprotonation of the gsh carboxyl groups with potassium t - butoxide, gsh - coated cdse / cdzns qds are easily dispersed in water. the particle size of gsh - qds (6.5 nm) and their fluorescence properties in phosphate buffer were similar to those of unmodified qds in chloroform (data not shown). gsh - qds were encapsulated in liposomes via the hydration of lipid film using a gsh - qd suspension. liposomes with different surface charges were examined ; the negatively charged liposomal constitution was dspc : dcp : chol (8 : 2 : 1), and the positively charged constitution was dspc : sa : chol (8 : 0.2 : 1). the fluorescence of gsh - qd - loaded liposomes was observed under a uv lamp for different liposomal compositions (figures 2(a) and 2(b)). the gsh - qds in the positively charged sa liposomes emitted strong fluorescence (figure 2(b)). in contrast, the signal in the dcp liposome was less intense (figure 2(a)). the gsh - qds were negatively charged because of the dissociation of the carboxyl group of gsh. these results suggested that the gsh - qds were present in the inner aqueous phase of the liposome and that they were adsorbed onto the sa lipid bilayer electrostatically, whereas the gsh - qds did not associate with the dcp lipid membrane of liposomes. as a result, gsh - qd - loaded sa liposomes displayed strong fluorescence compared with gsh - qd - loaded dcp liposomes. the dls particle size of gsh - qd - loaded sa liposomes (219.5 nm) was larger than that of unloaded (115.4 nm) and gsh - qd - loaded dcp liposomes (104.7 nm) because of the interaction of qds with sa on the liposomal surface. these considerations were supported by tem images of gsh - qd - loaded sa liposomes (figure 2(c)). gsh - qds were observed in the aqueous core as well as on the sa liposomal surface. we have developed several types of liposomes modified with mucoadhesive polymers such as chitosan (positively charged polymer) or carbopol (negatively charged polymer) as oral drug delivery systems. the liposome surface modification was achieved by simply mixing surface modifier solutions with negatively or positively charged (counter charged for surface modifier) core liposomes ; electrostatic adsorption onto the charged liposomes was spontaneous. therefore, we evaluated the cellular association of dcp (negative) and sa (positive) liposomes. the cellular uptake of the different qd - loaded liposomal preparations was evaluated visually using clsm (figures 3(a) and 3(b)). clsm revealed fluorescence activity in the caco-2 cells exposed to qd - loaded liposomes. in in vitro cytotoxicity tests (figure 3(c)), qd - labeled liposomes did not negatively affect the viability of caco-2 cells during the uptake experiments. the images shown are z - sections through the center of the cells, which indicated that the fluorescence observed was the result of liposomal localization inside the cells. the uptake fluorescence of dcp liposomes with a negative zeta potential and sa liposomes with a positive zeta potential could be observed in the cytosol after analyzing the caco-2 cells by clsm, suggesting that liposomes were internalized by the caco-2 cells. the uptake of sa liposomes into the caco-2 cells was higher than that of dcp liposomes. these results suggested that cationic sa on the surface of liposomes enhanced the association between sa liposomes and negatively charged cell membranes via electrostatic interactions and that this association might serve to increase cellular uptake. the potential toxic effects of qd - loaded liposomes were evaluated in vitro using caco-2 cells by the mts assay, which is a well - established technique for assessing toxicity (figure 3(c)). the viability of caco-2 cells was almost unchanged with exposure to qd - loaded liposomal suspensions in the range used in the cell studies that followed. the specific adhesion and uptake of qd - loaded liposomes into the intestinal mucosa were evaluated 2 h after oral administration. figure 4 shows the fluorescence signals in the mucosal tissues of intestinal specimens after oral administration of liposomes. the intestinal tubes were removed from the rats at the appropriate time after oral administration of the liposomes. the detectability of qd and gsh - qd - loaded liposomes in the intestinal tube was evaluated by observing the residual liposomes on the mucosa. small amounts of gsh - qds were observed (figure 4(b)), whereas large amounts of hydrophobic qds were detected by clsm (figure 4(a)). these results suggested that unmodified qd - loaded liposomes were better for tracing and detecting liposomes in the body after oral administration. we confirmed that the fluorescence activity of gsh - qds disappeared in an acidic ph buffer that mimicked gastric juice (data not shown). the dissociation of gsh from the qd surface and degradation of qds could be induced by gastric juice in the stomach. in contrast, qds embedded in the lipid bilayers were protected by the surrounding lipids from the acidic condition of the gut (figure 4(a)). therefore, gsh - qds were not suitable as labeling markers of orally administered liposomes. however, this mucoadhesive liposome system was investigated for other administration routes such as pulmonary and eye - drop administration [20, 21 ]. gsh - qds entrapped in liposomes appear to be effective for analyzing liposomal behavior in the body for administration routes that do not involve the stomach.. therefore, the combination of qds and gsh - qds might be effective for liposomal detection. in the present study, non - nir qd - loaded liposomes were orally administered to rats. the main purpose of this study was to evaluate whether orally administered qds could be used as fluorescence markers as a replacement for organic fluorescence materials. we consider this experiment useful for illustrating the tracing potential of qds for orally administered liposomes. further investigation is required to optimize the efficacy of nir - qds in noninvasive imaging for evaluating the pharmacodynamics of liposomes and the mucoadhesive effects of surface modifiers. in summary, we prepared two types of qd - loaded liposomes : unmodified (hydrophobic) qds embedded in the lipid bilayer and gsh - qds dispersed in the inner aqueous phase of liposomes. qd - loaded liposomes had high biocompatibility and low toxicity in caco-2 cells. using clsm, the fluorescent signal of qds in the liposomes could be detected in the intestinal mucosa after oral administration. these results suggested that qds are useful materials as tracers of liposomes in in vivo applications. | we have developed submicron - sized liposomes modified with a mucoadhesive polymer to enhance peptide drug absorption after oral administration. liposomal behavior in the gastrointestinal tract is a critical factor for effective peptide drug delivery. the purpose of this study was to prepare quantum dot- (qd-) loaded submicron - sized liposomes and examine liposomal behavior in the body after oral administration using in vivo fluorescence imaging. two types of cdse / cdzns qds with different surface properties were used : hydrophobic (unmodified) qds and hydrophilic qds with glutathione (gsh) surface modifications. qd- and gsh - qd - loaded liposomes were prepared by a thin film hydration method. transmission electron microscopy revealed that qds were embedded in the liposomal lipid bilayer. conversely, gsh - qds were present in the inner aqueous phase. some of the gsh - qds were electrostatically associated with the lipid membrane of stearylamine - bearing cationic liposomes. qd - loaded liposomes were detected in caco-2 cells after exposure to the liposomes, and these liposomes were not toxic to the caco-2 cells. furthermore, we evaluated the in vivo bioadhesion and intestinal penetration of orally administered qd - loaded liposomes by observing the intestinal segment using confocal laser scanning microscopy. |
genital infections with herpes simplex virus types 1 and 2 (hsv 1 and 2) are common, but infrequently cause noticeable symptoms. the seroprevalence of hsv-2 was 9.1% in 2000 for ontario (2) while a prevalence of 21.9% among individuals older than 12 years was reported in the united states between 19881994 (3). more recently (19992004), xu. a small proportion of infected individuals reported a diagnosis of genital herpes ; 9.9% in 19881994 and 14.3% in 19992004 (4). thus, most infected individuals are unaware that they are infected but are still able to spread the virus asymptomatically, serving as important transmitters of the virus. although hsv-1 typically causes oropharyngeal lesions and is usually transmitted by non - sexual contact during childhood (5), the virus can cause genital herpes when transmitted by vaginal or oral sex, often without visible lesions (58). the lesions of hsv-1 are clinically indistinguishable from those of hsv-2 (9). however, hsv-1 genital herpes infections are less severe and recur less often than hsv-2 (912). howard. (2) found a seroprevalence of 51.1% for hsv-1 in 2000 for ontario. similarly in the us, hsv-1 was widespread in 19992004 (57.7%), but the number of hsv-1 infected individuals reporting genital herpes was 1.8% in this time period (4). hsv-1 comprises 1015% of all genital herpes cases in the us, although regional variations exist (13). for instance, in an american university student population, the percentage of genital herpes due to hsv-1 increased from 30.9% in 1993 to 77.6% in 2001 (14). the high prevalence of genital infections due to both hsv-1 and hsv-2 suggests a need to reduce the transmission of genital herpes. in 2003, the us food and drug administration approved an indication for valtrex (valacyclovir) to reduce genital herpes transmission : valtrex reduces the risk of heterosexual transmission of genital herpes to susceptible partners with healthy immune systems when used as suppressive therapy in combination with safer sex practices. (15) the same indication was approved by health canada in 2004 (16). extensive marketing directed toward patients encourages them to have their hsv serostatus determined (and indirectly encourages valacyclovir use in discordant couples). both the positive and negative aspects associated with using valacyclovir suppressive therapy to prevent transmission in couples discordant for hsv will be discussed in this paper in an attempt to better inform decision - making. a literature review was performed to determine the costs and benefits of suppressive valacyclovir therapy. in addition, the cost of using valacyclovir to achieve a 30% reduction in the population incidence of hsv-2 genital herpes was calculated (table 1). data was used from previously published articles, and all costs were converted to 2000 us dollars. for this analysis, at such a high coverage, there would be fewer outbreaks among those receiving suppressive valacyclovir. these savings were conservatively calculated by multiplying the annual cost of genital herpes by the percentage of individuals assumed to have no recurrent symptoms as a result of receiving valacyclovir suppressive therapy. next, the lifetime savings from reducing the annual incidence of hsv-2 by 30% was calculated. the number of years required for these savings to balance the cost of the program was also determined. in addition, the cost (2000 us dollars) of preventing a single transmission of hsv-2 genital herpes by using suppressive valacyclovir therapy was calculated (table 2). although the number needed to treat to prevent one transmission varies among studies, the figure used in this analysis was considered a conservative estimate. the lifetime savings from preventing the single transmission was calculated using the larger lifetime cost to treat men. further, there would again be potential savings from a reduction in symptoms among those receiving suppressive valacyclovir therapy. it should also be noted that this simplified analysis is limited since it is based on data from heterogeneous sources, each with its own methodologies and assumptions. the primary benefit of using suppressive valacyclovir therapy is to reduce transmission within discordant couples. corey. (17) found that prescribing valacyclovir to individuals with symptomatic, recurrent hsv-2 infections the acquisition of genital herpes in their discordant partner was reduced from 3.6% in the placebo group to 1.9% in the valacyclovir group (hazard ratio 0.52 ; 95% confidence interval 0.270.99 ; p = 0.04). since the absolute reduction was not large, the yearly number needed to treat was 38. individuals in the valacyclovir group received 500 mg of valacyclovir once daily for eight months. this therapy effectively reduced viral shedding relative to the placebo group. as a result, it reduces transmission when the source partner does not have any recognizable symptoms but is still shedding virus, which is when most transmission events occur (18,19). the potential benefits of using suppressive valacyclovir to reduce transmissions are obvious : reducing the psychological and physical harms of acquiring genital herpes, while lowering the economic costs associated with this infection. psychologically, acquiring symptomatic genital herpes can have a significant emotional impact on patients (2022). such patients may suffer from social isolation and have difficulty initiating relationships (23,24). in contrast, other studies have shown that appropriate counseling can significantly reduce the psychological harm caused by diagnosing genital herpes (2527). receiving genital herpes additionally, the source partner may be significantly worried about transmitting genital herpes to their non - infected partner (23). therefore by reducing transmission, valacyclovir can diminish possible psychological harms, although counseling has been shown to be effective in this regard as well (26). potential physical harms due to genital herpes can be prevented with suppressive therapy, assuming that the partner acquires a recognizable, symptomatic infection. genital herpes caused by hsv-1 recurs infrequently and the frequency decreases over time with a recurrence duration of 7 days (12,28). in contrast, hsv-2 genital herpes recurs more frequently and the rate of recurrence decreases slowly over time with a recurrence duration of 8.5 - 10.1 days (2830). local symptoms for hsv-2 infection include pain, itching, and vaginal or urethral discharge (30). fever, headache, and malaise occur in 39% of men and 68% of females during primary infection (30). pain during recurrent genital herpes is reported as severe in 11% of patients, moderate in 36%, mild in 48%, and absent in 4% (21). complications of genital herpes include dysuria, aseptic meningitis, autonomic nervous system dysfunction, transverse myelitis, yeast infections, and extragenital lesions (30). economically, reducing the population incidence of genital herpes will decrease such healthcare costs as hospitalizations, clinical examinations, consultations, and tests. indirect economic costs for patients, such as time off work and traveling costs, will also be avoided (21,23,31,32). recurrent genital herpes can also lower work effectiveness during severe symptomatic events (21). (32) performed an analysis of the economic burden of genital herpes in the us for 1996. they estimated the cost of genital herpes to range from $ 283 million (0.1% of the us health care expenditure) to $ 984 million with indirect costs totaling an additional $ 214 million. of the total cost, 49.7% came from drug expenditures, 47.7% from medical care (consultations and lab testing), and 2.6% from hospital costs. szucs and colleagues (32) also calculated a cost of $ 60 000 per case of neonatal herpes and $ 2 500 for each cesarean section. in summary, reducing transmission will diminish the psychological, physical, and certain economic burdens of symptomatic genital herpes. however, it should be noted that only 14.3% of individuals with hsv-2 develop recognizable, symptomatic genital herpes (4), and as a result, the functional effect of reducing transmission on decreasing this burden is limited. there are numerous costs associated with suppressive valacyclovir therapy in couples discordant for genital herpes. condoms also protect against other sexually transmitted infections that an individual in the relationship may have. however, corey. (17) found a similar rate of condom use between the valacyclovir and placebo groups, and regardless of suppressive therapy, condom use remains low among discordant couples (19,33). it appears that suppressive valacyclovir therapy does not change couples sexual behaviours, but further studies are required to adequately explore this relationship. furthermore, it may be suggested that suppressive use of valacyclovir will cause antiviral resistance. despite a lack of long - term studies, no resistance to this drug has been found for therapy lasting one year (3537). in long - term studies with acyclovir, resistance is uncommon in immunocompromised individuals (~5%) and rare in immunocompetent individuals (38,39). currently, there appears to be little physical harm in taking valacyclovir every day for more than a year in healthy patients (3537). in patients infected with human immunodeficiency virus (hiv), there are no data on the safety of therapy lasting more than 6 months (40). headaches, nausea and vomiting, dizziness, and abdominal pain are the most commonly reported adverse effects. although patients with recurrent genital herpes prefer suppressive over episodic therapy (22), it is not known how asymptomatic patients would respond to and comply with daily therapy over the long - term. as previously described, there would be some financial savings with suppressive valacyclovir therapy, but there would also be significant economic costs associated with this program due to drug costs and screening (31,32,41). firstly, valacyclovir costs $ 118.47 for 30 tablets of 500 mg, totaling $ 1441 a year (canadian dollars, 2008). another significant cost would be identifying those individuals that have an asymptomatic or unrecognized clinical infection. according to corey (42), 60% of hsv-2 seropositive individuals have unrecognized symptoms, while 20% are subclinical (asymptomatic) with another 20% being recognized symptomatic. however all three groups have similar frequencies of asymptomatic shedding events (19), defined as detection of hsv on the surface of skin or mucosa in the absence of genital lesions (43). most individuals with unrecognized symptomatic genital herpes can identify their symptoms with counseling and education (42,44). additionally, type - specific serologic tests are capable of distinguishing between hsv-1 and hsv-2 (45). the presence of antibodies to hsv-1 alone gives no information on the presence of genital herpes because the site of the infection, oral or genital, can not be determined (26). since hsv-1 is very common, genital herpes due to hsv-1 would be difficult to detect without swabbing genital lesions. in addition, the frequency of testing required to identify individuals with genital herpes adds to the cost of screening (45). if testing were recommended with every new sexual partner, there would be considerable strain placed on the healthcare system and the patient. moreover, it is predicted that there would be significant psychological harm associated with diagnosing patients with genital herpes who were previously unaware of their infection. through a large screening program, physicians may actually cause more psychosocial harm to asymptomatic individuals, such as social isolation and reluctance to initiate relationships, than the physical harms these individuals experience (23,24,46,47). however as previously noted, studies have shown that counseling is effective in reducing psychological harm upon diagnosis (25,26). in addition, disclosing the problem to their current partner, who may raise questions of infidelity or past sexual history, may damage or ruin the relationship. due to the additional economic cost and psychological problems of screening the general population, (48) that is, a diagnosis would be more important in couples where transmission would be significantly harmful. such circumstances may include individuals who are at increased risk of hiv infections, hiv positive patients, and patients with a partner with genital herpes (48). by diagnosing these individuals with genital herpes current models suggest that suppressive therapy will have a minimal effect on reducing the population incidence of genital herpes at the currently low levels of diagnosis and treatment (50). the coverage level and the duration of suppressive therapy are important factors in reducing the population incidence (50). the coverage level is primarily determined by the proportion of patients diagnosed and the proportion of diagnosed that are receiving therapy. with a coverage level of 3.2% (the current coverage in the us) and 3 years of valacyclovir therapy, it is expected that there would be a 1.8% reduction in new cases after 5 years and a 2.8% reduction after 25 years (50). the percent reduction of new cases increases to 65% with a coverage of 60% after 25 years. according to williams. (50), a coverage rate of 60% is unrealistic, but they suggest that a coverage rate of 30% is theoretically possible and would reduce incidence by 30% after 25 years. additionally, the duration of suppressive therapy can be increased. at a coverage level of 3.2%, the incidence of hsv-2 infection would be expected to decrease by 3.5% after 25 years with 5 years of therapy, compared to 1.3% with a 1-year therapy program. the financial cost of administering suppressive valacyclovir over three years to 30% of americans with hsv-2 genital herpes is approximately $ 50.3 billion (table 1). by treating such a large number of patients with suppressive therapy, it is estimated that $ 816 million would be saved over the three years from reduced outbreaks among infected individuals. thus, the net cost of a three - year program is $ 49.5 billion. using the lifetime cost of an individual with genital herpes (51), the yearly savings from the 30% reduction in incidence is $ 540 million. therefore, the savings from the program would balance the cost after 92 years. obviously, one must also consider the increased quality of life gained from a decrease in incidence. a conservative estimate of the cost to prevent one hsv-2 transmission is $ 12,932 (table 2). this cost would outweigh the benefits gained since a cost of $ 8,200 per prevention translates to $ 140,000 per quality - adjusted - life - year gained, which is above the cost effective threshold of $ 50,000-$100,000 (52). moreover, these calculations do not include the screening costs that would be necessary to identify asymptomatic individuals and reach the 30% coverage rate. such screening would be essential to this program because they are the majority of individuals with genital herpes and the main group transmitting the virus (42,46). the compliance may also be lower in the general public than in the trial performed by corey. (17), especially among asymptomatic or unrecognized patients (50). at an individual level in canada, the financial costs would be that of valacyclovir, $ 1441 each year, and the cost of screening to determine whether individuals are infected and if couples are discordant. the psychological and physical benefits noted above may convince a symptomatic, discordant couple into asking for valacyclovir to reduce the likelihood of transmission, but in how many instances will valacyclovir reduce transmission ? corey. (17) found a 48% reduction in the risk of transmission, but this reduction was from an acquisition risk of 3.6% to 1.9%. furthermore, when transmission does occur, most people do not develop symptomatic genital herpes. it is essential that patients understand these facts in order to make an informed decision. these factors must be discussed before a discordant couple makes a decision regarding suppressive valacyclovir therapy. for example, prescription may depend on the hsv-1 serostatus of the partner without genital herpes. if this individual has hsv-1, they have a lower probability of developing symptomatic genital herpes upon acquiring hsv-2 (30,5355). in addition, the virus type (hsv-1 or hsv-2) of the source partner may be important to determine because each type causes different symptoms and has different modes of transmission. type 1 (genital) is less likely to cause recurrent symptoms for the partner that acquires symptomatic infection (912). compared to hsv-2, there is less asymptomatic shedding and, therefore, a reduced transmission rate with type 1 genital herpes (11). in addition, oral sex from a partner with a history of oral herpes is a risk factor for hsv-1 transmission (68,56). transmission can occur without lesions because virus in the oropharynx can shed asymptomatically (56). transmission from oral sex is less likely for hsv-2 because this virus is not as likely to cause an infection in the oropharynx (7,13). if an individual has many recurrences each year, then it would be more feasible to prescribe suppressive therapy because the individual will receive the added benefit of reduced symptoms. however, suppressive valacyclovir therapy is not typically necessary for hsv-1 genital herpes (12,57), and the effect of valacyclovir on hsv-1 transmission has not been adequately explored. nevertheless, valacyclovir does decrease the presence of the virus in saliva (58) and has been examined in transmission between wrestlers (59). another factor to consider is the duration of the infection in the source partner because infectivity likely decreases with time due to a decrease in shedding (11,33). (49) found the mean number of sex acts before transmission was 40 while mertz. for hsv-2, asymptomatic shedding was three fold more frequent during the first three months after resolution of primary infection than after these three months (11). it is also important to discuss the number of sexual partners and the nature of the relationship. between 19992004, the seroprevalence of hsv-2 was 39.9% in individuals who had 50 or more lifetime partners compared to 3.8% who had one lifetime partner (4). transmitting genital herpes may increase the receptive partner s risk of contracting hiv in the future. the risk of acquiring hiv is approximately three times greater in hsv-2 infected men or women (60). the suggested mechanism for this interaction is likely due to the destruction of the epithelial layer by hsv-2 and attraction of cd4-positive cells (61). this relationship is important for partners that may have a higher risk of contracting hiv due to location, drug use, or sexual behaviours. additionally, special consideration should be given to a hiv positive, hsv-2 negative partner because hsv-2 reactivation correlates with increased plasma hiv-1 levels (62,63). hsv-2 can increase morbidity and mortality for hiv infected people and may accelerate the course of hiv disease (64). hsv-2 positive, hiv-1 positive individuals may also be more efficient at transmitting hiv than hsv-2 negative individuals (64). thus, it is important to prevent transmission of hsv-2 to hiv positive individuals (65,66). the interaction between hsv-1 and hiv has yet to be adequately explored (67). couples involving a hsv - negative pregnant woman and a hsv positive partner should be made aware that vertical transmission (genital hsv-1 and hsv-2) can be serious for infants, causing death or neurological impairment from disseminated infection of multiple organs and the central nervous system (47,68). disease of the skin, eyes, or mouth can also occur (68). however, the highest risk of neonatal injury occurs when the pregnant woman acquires either virus type near the time of labor (69). the emotional and financial costs of vertical hsv transmission are obviously significant (31). the duration of the relationship is important, with an 8-fold risk in transmitting hsv-2 in relationships of one year or less compared to relationships existing for more than one year (8). besides suppressive valacyclovir therapy, counseling, condom use, and abstinence are strategies that should reduce the risk of transmission. thus, such individuals entering relationships with hsv positive partners may benefit from suppressive therapy for the source partner. finally, it is important to consider the degree of emotional stress in the source partner regarding transmission and the emotional problems that would likely occur in the non - infected partner upon acquiring genital herpes (71). some individuals may have a more negative stigma regarding genital herpes than others and acquiring it would be severely damaging to these patients. nevertheless, some couples relationships may be severely affected by this disease and thus may require suppressive valacyclovir therapy more than other couples. the primary benefit of using suppressive valacyclovir therapy is to reduce transmission within discordant couples. corey. (17) found that prescribing valacyclovir to individuals with symptomatic, recurrent hsv-2 infections the acquisition of genital herpes in their discordant partner was reduced from 3.6% in the placebo group to 1.9% in the valacyclovir group (hazard ratio 0.52 ; 95% confidence interval 0.270.99 ; p = 0.04). since the absolute reduction was not large, the yearly number needed to treat was 38. individuals in the valacyclovir group received 500 mg of valacyclovir once daily for eight months. this therapy effectively reduced viral shedding relative to the placebo group. as a result, it reduces transmission when the source partner does not have any recognizable symptoms but is still shedding virus, which is when most transmission events occur (18,19). the potential benefits of using suppressive valacyclovir to reduce transmissions are obvious : reducing the psychological and physical harms of acquiring genital herpes, while lowering the economic costs associated with this infection. psychologically, acquiring symptomatic genital herpes can have a significant emotional impact on patients (2022). such patients may suffer from social isolation and have difficulty initiating relationships (23,24). in contrast, other studies have shown that appropriate counseling can significantly reduce the psychological harm caused by diagnosing genital herpes (2527). receiving genital herpes additionally, the source partner may be significantly worried about transmitting genital herpes to their non - infected partner (23). therefore by reducing transmission, valacyclovir can diminish possible psychological harms, although counseling has been shown to be effective in this regard as well (26). potential physical harms due to genital herpes can be prevented with suppressive therapy, assuming that the partner acquires a recognizable, symptomatic infection. genital herpes caused by hsv-1 recurs infrequently and the frequency decreases over time with a recurrence duration of 7 days (12,28). in contrast, hsv-2 genital herpes recurs more frequently and the rate of recurrence decreases slowly over time with a recurrence duration of 8.5 - 10.1 days (2830). local symptoms for hsv-2 infection include pain, itching, and vaginal or urethral discharge (30). fever, headache, and malaise occur in 39% of men and 68% of females during primary infection (30). pain during recurrent genital herpes is reported as severe in 11% of patients, moderate in 36%, mild in 48%, and absent in 4% (21). complications of genital herpes include dysuria, aseptic meningitis, autonomic nervous system dysfunction, transverse myelitis, yeast infections, and extragenital lesions (30). economically, reducing the population incidence of genital herpes will decrease such healthcare costs as hospitalizations, clinical examinations, consultations, and tests. indirect economic costs for patients, such as time off work and traveling costs, will also be avoided (21,23,31,32). recurrent genital herpes can also lower work effectiveness during severe symptomatic events (21). (32) performed an analysis of the economic burden of genital herpes in the us for 1996. they estimated the cost of genital herpes to range from $ 283 million (0.1% of the us health care expenditure) to $ 984 million with indirect costs totaling an additional $ 214 million. of the total cost, 49.7% came from drug expenditures, 47.7% from medical care (consultations and lab testing), and 2.6% from hospital costs. szucs and colleagues (32) also calculated a cost of $ 60 000 per case of neonatal herpes and $ 2 500 for each cesarean section. in summary, reducing transmission will diminish the psychological, physical, and certain economic burdens of symptomatic genital herpes. however, it should be noted that only 14.3% of individuals with hsv-2 develop recognizable, symptomatic genital herpes (4), and as a result, the functional effect of reducing transmission on decreasing this burden is limited. there are numerous costs associated with suppressive valacyclovir therapy in couples discordant for genital herpes. condoms also protect against other sexually transmitted infections that an individual in the relationship may have. however, corey. (17) found a similar rate of condom use between the valacyclovir and placebo groups, and regardless of suppressive therapy, condom use remains low among discordant couples (19,33). it appears that suppressive valacyclovir therapy does not change couples sexual behaviours, but further studies are required to adequately explore this relationship. furthermore, it may be suggested that suppressive use of valacyclovir will cause antiviral resistance. despite a lack of long - term studies, no resistance to this drug has been found for therapy lasting one year (3537). in long - term studies with acyclovir, resistance is uncommon in immunocompromised individuals (~5%) and rare in immunocompetent individuals (38,39). currently, there appears to be little physical harm in taking valacyclovir every day for more than a year in healthy patients (3537). in patients infected with human immunodeficiency virus (hiv), there are no data on the safety of therapy lasting more than 6 months (40). headaches, nausea and vomiting, dizziness, and abdominal pain are the most commonly reported adverse effects. although patients with recurrent genital herpes prefer suppressive over episodic therapy (22), it is not known how asymptomatic patients would respond to and comply with daily therapy over the long - term. as previously described, there would be some financial savings with suppressive valacyclovir therapy, but there would also be significant economic costs associated with this program due to drug costs and screening (31,32,41). firstly, valacyclovir costs $ 118.47 for 30 tablets of 500 mg, totaling $ 1441 a year (canadian dollars, 2008). another significant cost would be identifying those individuals that have an asymptomatic or unrecognized clinical infection. according to corey (42), 60% of hsv-2 seropositive individuals have unrecognized symptoms, while 20% are subclinical (asymptomatic) with another 20% being recognized symptomatic. however all three groups have similar frequencies of asymptomatic shedding events (19), defined as detection of hsv on the surface of skin or mucosa in the absence of genital lesions (43). most individuals with unrecognized symptomatic genital herpes can identify their symptoms with counseling and education (42,44). additionally, type - specific serologic tests are capable of distinguishing between hsv-1 and hsv-2 (45). the presence of antibodies to hsv-1 alone gives no information on the presence of genital herpes because the site of the infection, oral or genital, can not be determined (26). since hsv-1 is very common, genital herpes due to hsv-1 would be difficult to detect without swabbing genital lesions. in addition, the frequency of testing required to identify individuals with genital herpes adds to the cost of screening (45). if testing were recommended with every new sexual partner, there would be considerable strain placed on the healthcare system and the patient. moreover, it is predicted that there would be significant psychological harm associated with diagnosing patients with genital herpes who were previously unaware of their infection. through a large screening program, physicians may actually cause more psychosocial harm to asymptomatic individuals, such as social isolation and reluctance to initiate relationships, than the physical harms these individuals experience (23,24,46,47). however as previously noted, studies have shown that counseling is effective in reducing psychological harm upon diagnosis (25,26). in addition, disclosing the problem to their current partner, who may raise questions of infidelity or past sexual history, may damage or ruin the relationship. due to the additional economic cost and psychological problems of screening the general population, screening of targeted populations, however, may be appropriate. (48) that is, a diagnosis would be more important in couples where transmission would be significantly harmful. such circumstances may include individuals who are at increased risk of hiv infections, hiv positive patients, and patients with a partner with genital herpes (48). by diagnosing these individuals with genital herpes, infected individuals may recognize outbreaks and abstain during such periods. current models suggest that suppressive therapy will have a minimal effect on reducing the population incidence of genital herpes at the currently low levels of diagnosis and treatment (50). the coverage level and the duration of suppressive therapy are important factors in reducing the population incidence (50). the coverage level is primarily determined by the proportion of patients diagnosed and the proportion of diagnosed that are receiving therapy. with a coverage level of 3.2% (the current coverage in the us) and 3 years of valacyclovir therapy, it is expected that there would be a 1.8% reduction in new cases after 5 years and a 2.8% reduction after 25 years (50). the percent reduction of new cases increases to 65% with a coverage of 60% after 25 years. according to williams. (50), a coverage rate of 60% is unrealistic, but they suggest that a coverage rate of 30% is theoretically possible and would reduce incidence by 30% after 25 years. additionally, the duration of suppressive therapy can be increased. at a coverage level of 3.2%, the incidence of hsv-2 infection would be expected to decrease by 3.5% after 25 years with 5 years of therapy, compared to 1.3% with a 1-year therapy program. the financial cost of administering suppressive valacyclovir over three years to 30% of americans with hsv-2 genital herpes is approximately $ 50.3 billion (table 1). by treating such a large number of patients with suppressive therapy, it is estimated that $ 816 million would be saved over the three years from reduced outbreaks among infected individuals. thus, the net cost of a three - year program is $ 49.5 billion. using the lifetime cost of an individual with genital herpes (51), obviously, one must also consider the increased quality of life gained from a decrease in incidence. a conservative estimate of the cost to prevent one hsv-2 transmission is $ 12,932 (table 2). this cost would outweigh the benefits gained since a cost of $ 8,200 per prevention translates to $ 140,000 per quality - adjusted - life - year gained, which is above the cost effective threshold of $ 50,000-$100,000 (52). moreover, these calculations do not include the screening costs that would be necessary to identify asymptomatic individuals and reach the 30% coverage rate. such screening would be essential to this program because they are the majority of individuals with genital herpes and the main group transmitting the virus (42,46). the compliance may also be lower in the general public than in the trial performed by corey. at an individual level in canada, the financial costs would be that of valacyclovir, $ 1441 each year, and the cost of screening to determine whether individuals are infected and if couples are discordant. the psychological and physical benefits noted above may convince a symptomatic, discordant couple into asking for valacyclovir to reduce the likelihood of transmission, but in how many instances will valacyclovir reduce transmission ? corey. (17) found a 48% reduction in the risk of transmission, but this reduction was from an acquisition risk of 3.6% to 1.9%. furthermore, when transmission does occur, most people do not develop symptomatic genital herpes. it is essential that patients understand these facts in order to make an informed decision. these factors must be discussed before a discordant couple makes a decision regarding suppressive valacyclovir therapy. for example, prescription may depend on the hsv-1 serostatus of the partner without genital herpes. if this individual has hsv-1, they have a lower probability of developing symptomatic genital herpes upon acquiring hsv-2 (30,5355). in addition, the virus type (hsv-1 or hsv-2) of the source partner may be important to determine because each type causes different symptoms and has different modes of transmission. type 1 (genital) is less likely to cause recurrent symptoms for the partner that acquires symptomatic infection (912). compared to hsv-2, there is less asymptomatic shedding and, therefore, a reduced transmission rate with type 1 genital herpes (11). in addition, oral sex from a partner with a history of oral herpes is a risk factor for hsv-1 transmission (68,56). transmission can occur without lesions because virus in the oropharynx can shed asymptomatically (56). transmission from oral sex is less likely for hsv-2 because this virus is not as likely to cause an infection in the oropharynx (7,13). if an individual has many recurrences each year, then it would be more feasible to prescribe suppressive therapy because the individual will receive the added benefit of reduced symptoms. however, suppressive valacyclovir therapy is not typically necessary for hsv-1 genital herpes (12,57), and the effect of valacyclovir on hsv-1 transmission has not been adequately explored. nevertheless, valacyclovir does decrease the presence of the virus in saliva (58) and has been examined in transmission between wrestlers (59). another factor to consider is the duration of the infection in the source partner because infectivity likely decreases with time due to a decrease in shedding (11,33). (49) found the mean number of sex acts before transmission was 40 while mertz. for hsv-2, asymptomatic shedding was three fold more frequent during the first three months after resolution of primary infection than after these three months (11). it is also important to discuss the number of sexual partners and the nature of the relationship. between 19992004, the seroprevalence of hsv-2 was 39.9% in individuals who had 50 or more lifetime partners compared to 3.8% who had one lifetime partner (4). transmitting genital herpes may increase the receptive partner s risk of contracting hiv in the future. the risk of acquiring hiv is approximately three times greater in hsv-2 infected men or women (60). the suggested mechanism for this interaction is likely due to the destruction of the epithelial layer by hsv-2 and attraction of cd4-positive cells (61). this relationship is important for partners that may have a higher risk of contracting hiv due to location, drug use, or sexual behaviours. additionally, special consideration should be given to a hiv positive, hsv-2 negative partner because hsv-2 reactivation correlates with increased plasma hiv-1 levels (62,63). hsv-2 can increase morbidity and mortality for hiv infected people and may accelerate the course of hiv disease (64). hsv-2 positive, hiv-1 positive individuals may also be more efficient at transmitting hiv than hsv-2 negative individuals (64). thus, it is important to prevent transmission of hsv-2 to hiv positive individuals (65,66). the interaction between hsv-1 and hiv has yet to be adequately explored (67). couples involving a hsv - negative pregnant woman and a hsv positive partner should be made aware that vertical transmission (genital hsv-1 and hsv-2) can be serious for infants, causing death or neurological impairment from disseminated infection of multiple organs and the central nervous system (47,68). disease of the skin, eyes, or mouth can also occur (68). however, the highest risk of neonatal injury occurs when the pregnant woman acquires either virus type near the time of labor (69). the emotional and financial costs of vertical hsv transmission are obviously significant (31). the duration of the relationship is important, with an 8-fold risk in transmitting hsv-2 in relationships of one year or less compared to relationships existing for more than one year (8). besides suppressive valacyclovir therapy, counseling, condom use, and abstinence are strategies that should reduce the risk of transmission. thus, such individuals entering relationships with hsv positive partners may benefit from suppressive therapy for the source partner. finally, it is important to consider the degree of emotional stress in the source partner regarding transmission and the emotional problems that would likely occur in the non - infected partner upon acquiring genital herpes (71). some individuals may have a more negative stigma regarding genital herpes than others and acquiring it would be severely damaging to these patients. nevertheless, some couples relationships may be severely affected by this disease and thus may require suppressive valacyclovir therapy more than other couples. the high prevalence of genital infections with hsv-1 and hsv-2 is of great concern and indicates that a solution is required. nevertheless, suppressive valacyclovir therapy is not a feasible method of reducing the incidence of genital herpes because of the overwhelming economic cost and issues of identifying asymptomatic individuals. furthermore, the stigma and fear associated with genital herpes only increases the need to educate patients. if a symptomatic individual desires to take suppressive therapy, the physician must inform the patient about the influence of valacyclovir on transmission along with the probability that their partner will become symptomatic. moreover, patients should consider certain factors that increase the importance of transmission when making a decision, such as the serostatus of the non - infected individual, type of hsv infection, duration of infection, hiv risk, hiv serostatus, pregnancy, and likely degree of resultant emotional stress. finally, patients need to know about the realistic clinical harms of hsv infections. after better understanding the disease by discussing the above points, patients will be better positioned to make an informed decision regarding the use of prophylactic therapies like valacyclovir. | nocturnal asthma (na) is increasing in prevalence, affecting millions of people genital herpes is a widespread sexually transmitted infection caused by the herpes simplex viruses (hsv). suppressive valacyclovir therapy has been shown to significantly reduce hsv transmission. the benefits and costs of using valacyclovir to reduce transmission in couples discordant for genital herpes will be analyzed in order to better inform decision - making. by reducing transmission, the physical and psychological harms of living with symptomatic genital herpes will be prevented while saving on certain healthcare costs. however, the large number needed to treat and the low symptomatic rate among infected individuals may outweigh these benefits. the costs of trying to achieve a significant reduction in incidence include the psychological harms of identifying asymptomatic individuals through a large screening program and the economic costs of the antiviral agent and screening. when these issues are weighed, the high economic costs render a program to reduce incidence unfeasible. nevertheless, it is clinically important to consider the consequences of transmission at an individual level. the specific circumstances that influence the decision to use suppressive therapy are identified. |
cryptosporidium spp. are ubiquitous intracellular protozoan parasites that affect wide range of vertebrates like human and livestock. oocysts are infective stage of life cycle released via feces from infected hosts to environment in large amount. they are resistant to various environmental alterations and remain infective for long period in appropriate condition like surface of water and moist soils ; thus, they are liable for transmission of disease to animals and humans [2, 3 ]. cryptosporidium as an opportunistic agent infects the alimentary tract of hosts and have wide spectrum of clinical symptoms in human ranging from self - limiting and asymptomatic in immunocompetent individuals to severe and life - threatening in immunocompromised persons [2, 4, 5 ]. for first time, human cryptosporidiosis was diagnosed in individuals who had severe watery diarrhea during 1970s. during past decades, numerous evidences have confirmed that small mammals such as rodents are considered as carrier or reservoir for several infectious agents. also role of rodents in transferring the helminth and protozoan infections are clear [710 ]. surveys in different zones of iran indicate the existence of various rodent species such as the house mice (mus musculus), the black rat (rattus rattus), the brown rat (rattus norvegicus), and the himalayan rat (rattus pectoris) in country [8, 10, 11 ]. based on our previous survey in ahvaz district, southwest of iran, rodents could be considered as potential source of toxoplasma gondii infection for definitive hosts. small subunit - rrna (ssu 18s rrna) gene is being utilized for identification of cryptosporidium spp. according to ssu - rrna gene sequencing, at least 20 cryptosporidium species have been identified and more than sixty cryptosporidium genotypes have undeterminate status till now. approximately eight cryptosporidium species / genotypes including c. parvum, c. hominis, c. felis, c. meleagridis, c. ubiquitum, c. viatorum, c. canis, and c. cuniculus are the main species responsible for human infections, although c. hominis and c. parvum account for over 90% of human cryptosporidiosis worldwide [1, 2 ]. numerous epidemiological surveys have been performed throughout the globe and prevalence of cryptosporidium infection in rodents was highly varied from 63% in uk, 32.8% in united states of america (usa), 24.3% in italy, 7.6% in maryland, 11.5% in china, 25.8% in philippines, 8.2% in northern australia, 0% in northeast of iran (mashhad city), and 27.3% in north of iran (tehran city). due to lack of reports about cryptosporidium infection in rodents of southwest of iran till now, current study was aimed to determine the prevalence and molecular characterization of cryptosporidium spp. in this region. ahvaz city, capital of khuzestan province which is located in the southwest of iran (3150 n and 49 11 e), is ranked as the 7th largest city throughout the country and based on the latest census, its population was calculated at 1,395,184 in 352,128 families. weather temperature is highly variable throughout the year so that in summer temperature exceeds 50c whereas in winter it falls to 5c. also, annual average rainfall is approximately 230 mm. there is high density of rodents species and rat - man - domestic animals adjacency is remarkable in ahvaz city [9, 11 ]., mostly preyed on by cats and dogs, and, hence, could spread parasitic infections via other animals. ahvaz city initially divided into five geographical locations (north, west, south, east, and center). in each location sherman live traps were placed outdoor at the entrance of rodent colonies and baited with favorite piece of foods (including cucumber pieces, tomato, and roasted almonds). overall, 100 rodents were collected from three different species (6 m. musculus, 73 r. norvegicus, and 21 r. rattus). eventually trapped rodents were gathered and transferred to department of medical parasitology of ahvaz jundishapuruniversity of medical sciences. the trapped rodents were anaesthetized by putting the live traps in a thick transparent polythene bag and then a cotton swab was soaked in ether and placed near their nose. afterwards, the anaesthetized rodents were dissected and fecal samples gathered from rectum or large intestinal section. after sugar flotation (sg 1.266 ; 128 g sucrose and 100 distilled water) and modified ziehl - neelsen staining, the samples were examined to find the cryptosporidium spp. finally the samples were maintained at 2.5% potassium dichromate (k2cr2o7) and kept in refrigerator (1 - 2 weeks) until dna was extracted. dna extraction procedure was performed using qiaamp dna stool mini kit (qiaamp dna stool mini kit, usa) based on the manufacturer 's guideline. the extracted dna was kept in 20c for next tests. for nested - pcr method, we used two specific primers to detect 18s rrna gene whose length of produced fragments was 1325 bp and 830 bp. this dual stages technique was run using different primers that primary and secondary stages primers were as following : [4, 20, 23, 24 ]. first stage primers forward (f1) : 5-ttctagagctaatacatgcg-3reverse (r1) : 5-cccatttccttcgaaacagga-3 forward (f1) : 5-ttctagagctaatacatgcg-3 reverse (r1) : 5-cccatttccttcgaaacagga-3 second stage primers forward (f2) : 5-ggaagggttgtatttattagataaag-3reverse (r2) : 5-ctcataaggtgctgaaggagta-3finally pcr products after loading on 1.5% agarose gel and electrophoresis for 1.5 hours were stained with ethidium bromide and then visualized under uv light using gel doc device (uvidoc, gel documentation system, cambridge, uk). forward (f2) : 5-ggaagggttgtatttattagataaag-3 reverse (r2) : 5-ctcataaggtgctgaaggagta-3 rflp assay was done to determine the cryptosporidium spp. sspi and vspi endonuclease enzymes were employed for species recognition and genotyping, respectively, based on manufacturer 's protocol as earlier described. eventually visualization of the digested products was carried out under uv transilluminator after 1.5% agarose gel electrophoresis and ethidium bromide staining [20, 26 ]. the nested - pcr positive samples were purified using bioneer kit corporation (korea) and were sequenced by the same corporation. nucleotide sequence data reported in current article are available in the genbank at accession numbers ab986579, ab986580, and ab986581. three out of 100 samples were detected as positive for cryptosporidium spp. using modified ziehl - neelsen staining under light microscope. in addition, nested - pcr was done and showed 830 bp band which confirmed only 3 samples as cryptosporidium spp. so, overall prevalence of cryptosporidium infection in rodents of ahvaz city was calculated at 3% using both methods. all positive samples belonged to r. norvegicus (3/73) and from m. musculus (0/6) and r. rattus (0/21) no positive cases were observed. in order to determine the genotype, pcr - rflp technique using sspi and vspi restriction enzymes was utilized. with sspi restriction enzyme, three cuttings were seen in locations of 108, 258, and 421 bp visible on agarose gel after electrophoresis. also after using from vspi enzyme, three cuttings happened in locations 104, 106, and 600 bp (figure 2) and indicate c. parvum pattern. the amplified 18s rrna genes from pcr - rflp products of three c. parvum were sequenced. after submitting the results to the ddbj / genbank at accession numbers ab986579, ab986580, and ab986581, the nucleotide sequences were aligned with nucleotide sequences of c. parvum certified in genbank with accession number ab986578 (figure 3). based on findings, c. parvum was recognized as infectious agent of ahvaz rodents. rodents with maintaining the pathogens transmission cycle in surrounding regions play a key role in morbidity and mortality of human and livestock especially in areas with dense population. the routine method for diagnosis of cryptosporidium spp. is based on direct observation of oocysts in stool specimens using optical microscope. since this method has low sensitivity and needs expertise, also it is unable to distinguish between different species of parasites ; thus, molecular techniques like pcr have been used and developed recently. pcr - based techniques with high sensitivity, specificity, and rapidly features are capable of differentiating among species and genotypes in different specimens, that is, water, stool, and animal or human tissues, although they are expensive. previously, genotyping of cryptosporidium spp. has been done successfully by nested pcr - rflp method according to ssu - rrna (18s rrna) gene [4, 18, 20, 24, 26, 27 ]. according to previous epidemiological reports, prevalence and rate of infection of cryptosporidium spp. in rodents ranged from 0% to 63% and present investigation is the first report which focused on the prevalence and molecular detection of cryptosporidium spp. in wild rodents of ahvaz city, southwest of iran. we found 3% (3/100) prevalence by both direct microscopic observations with ziehl - neelsen staining and nested - pcr (830 bp), while, in bahrami. survey in tehran city (capital of iran) using these methods, the prevalence was reported at 13% and 27.3%, respectively. also all positive samples identified c. parvum by pcr - rflp using sspi and vspi restriction enzymes and were confirmed by sequencing that is in agreement with our study. differences between tehran and ahvaz findings could be justified with sample size, location of sampling, cities population, hygienic conditions, sewage systems, and so forth. it is worth mentioning that tehran as capital of iran is the most crowded city with highest density population over the country which is multifold than ahvaz. in mashhad city (northeast of iran), prevalence of cryptosporidium spp. with 0% was lower than our study (3%), while higher prevalence was reported from usa 32.8%, china 11.5%, australia 8.2%, philippines 25.8%, and tehran city (north of iran) 27.3%. based on current study, 18s rrna gene of c. parvum was detected only in r. norvegicus (3/73) and from r. rattus and m. musculus no positive samples were isolated. it should be mentioned that r. norvegicus is the most abundant rodent in southwest of iran (73/100) and highest prevalence and rate of infection (4.1%, 3/73) allocate to this species which corresponds to previous investigations [9, 11 ]. the obtained results of amplifying 18s rrna gene after sequencing were submitted to the ddbj / genbank under accession numbers ab986579, ab986580, and ab986581 and then compared with ab986578 as control. lv and colleagues studied the wild, laboratory, and pet rodents (totally 723 rodents from 18 species) in china and prevalence of cryptosporidium spp. was reported at 11.5% and c. parvum, c. muris, c. andersoni, and c. wrairi were identified, as well. prevalence in wild, laboratory, and pet rodents was 6.8%, 1.9%, and 21.8%, respectively. in another survey by ng - hublin. on 194 wild rats and mice from five species including the asian house rat (r. tanezumi), the rice - field rat (r. argentiventer), the pacific rat (r. exulans), r. norvegicus, and m. musculus in philippines, overall prevalence was reported at 25.8%. in addition, based on sequencing and phylogenetic analysis of 18s rrna gene and actin locus, c. muris, c. parvum, c. scrofarum, c. suis - like genotype, and rat genotypes i iv were recognized. in current research, only c. parvum was identified using 18s rrna gene sequencing. throughout the world c. parvum have been isolated from numerous animals such as calves or cattle, some ruminants (goats and sheep), horses, pigs, alpacas, some carnivores (gray wolves and dogs) [32, 33 ], reptiles, and rodents (rat, hamster, mice, nutria, chipmunk, squirrel, and capybara) [13, 16, 17, 20, 3437 ]. in addition predominant cryptosporidium species in iran is c. parvum that has been verified frequently, for example, 73.3% in humans and animals, 83.3% in tehran (human samples), 100% in rodents of tehran, 100% in cattle of ilam, and 68.8% in ahvaz (in immunocompromised patients and children). in rafiei investigation in southwest of iran on immunocompromised patients and children (kidney transplant recipients, persons with hematological malignancies, hiv+ patients, and children less than 5 years old), 390 stool specimens were collected and examined. moreover, 3 different species were identified using pcr - rflp based on 18s rrna gene including 11 c. parvum, 4 c. hominis, and 1 c. meleagridis which was in consistent with our results. according to rafiei survey and our study, c. parvum was identified as the most common species in both rodents and individuals in ahvaz city. in past studies c. parvum transmission from rodents to human the present investigation was based on sampling of limited rodents species in limited areas. in future, for better understanding of exact burden of cryptosporidium spp. and genetic diversity, studies should be designed on wide spectrum of both wild and pet rodents (rats, hamsters, mice, rabbits, etc.) in vast regions like throughout the khuzestan province. present paper was the first report which focused on the prevalence and molecular characterization of cryptosporidium spp. in wild rodents of ahvaz city, southwest of iran ; that showed rate of infection in r. norvegicus is remarkable. the results can help public health care to pursue new strategies (environmental sanitation, increasing the hygienic condition, increasing the hygienic condition, etc.). in future, adopting a suitable strategy for control and combat with rodents in order to decrease human cases is necessary and should be continued. | background. rodents could act as reservoir for cryptosporidium spp. specially c. parvum, a zoonotic agent responsible for human infections. since there is no information about cryptosporidium infection in rodents of ahvaz city, southwest of iran, hence, this survey was performed to determine the prevalence and molecular characterization of cryptosporidium spp. in this region. materials and methods. one hundred rodents were trapped from different regions of ahvaz city. intestine contents and fecal specimens of rodents were studied using both microscopy examination to identify oocyst and nested - polymerase chain reaction (pcr) technique for 18s rrna gene detection. eventually restriction fragment length polymorphism (rflp) method using sspi and vspi restriction enzymes was carried out to genotype the species and then obtained results were sequenced. results. three out of 100 samples were diagnosed as positive and overall prevalence of cryptosporidium spp. was 3% using both modified ziehl - neelsen staining under light microscope and nested - pcr (830 bp) methods. afterwards, pcr - rflp was performed on positive samples and c. parvum pattern was identified. finally pcr - rflp findings were sequenced and presence of c. parvum was confirmed again. conclusions. our study showed rodents could be potential reservoir for c. parvum. so an integrated program for control and combat with them should be adopted and continued. |
the basic method of treatment for chronic low back pain is conservative treatment, consisting of pharmacotherapy, kinesiotherapy, and physical therapy treatments. dynamic development of biomedical engineering results in new technical solutions applied in creating new medical devices. the devices that are currently used in physical therapy treatments support, and at times even replace, pharmacological treatment methods. in addition, due to their rare adverse effects, physical methods shorten treatment period, improve quality of life, and reduce therapy costs. one of the physical treatments most frequently used in back pain syndromes is electrical therapy. the main goal of using electrical stimulation in treating low back pain syndromes is an attempt to alleviate both pain and inflammation, as well as to reduce muscle tension in the affected regions [13 ]. however, on the basis of analysis of the available literature on electrotherapeutical methods of low back pain treatment, most studies fail to meet the basic criteria of evidence - based physiotherapy, which makes it extremely difficult to carry out a clear and objective analysis of clinical efficacy of treatments that are widely used in everyday practice. in the currently available research publications on electrical therapy of low back pain, generally no control groups or detailed randomization were used, and such studies were often conducted with relatively small groups of patients, based solely on subjective questionnaires and pain assessment scales (lacking measurement methods to objectify the therapeutic progress). the available literature also lacks any comprehensive and large - scale clinical study analyzing clinical efficacy of the numerous electrotherapeutic methods popular in physiotherapy in a single study, with consistent statistical calculations, which would allow for a reliable assessment of the studied treatments and their comparison to one another, and in relation to a representative control group. therefore, the aim of this study was to assess the effects of treating low back pain using selected electrical therapies. the study assesses the influence of individual electrotherapeutic treatments on reduction of pain, improvement of the range of movement in lower section of the spine, and improvement of motor functions and mobility. the research project was approved by the bioethics commission of the academy of physical education in katowice (no. the study included patients with low back pain, referred for physical therapy to the clinical research lab of the public higher medical professional school in opole, poland. qualification of patients was made by a team composed of an orthopedist, a neurologist, a neurosurgeon, an internist, a radiologist, and a physiotherapist. patients qualified for the study suffered from the l5-s1 discopathy, chronic radiating pain, and pseudoradicular syndrome, and had no previous surgical procedures within the spinal area. the patients were at least 18 years old and had valid certificates of the mri examination, confirming the diagnosis of low back pain syndrome (at least type iii changes in accordance with the modic classification in the l5s1 spine section). on the other hand, the allocation of patients who positively underwent the qualification procedure to specific groups was random (based on a computer number generator). exclusion criteria referred to patients who were diagnosed with : acute pain (less than 6 months), the radicular syndrome, discopathy of other spine regions (patients with early type i and ii changes were not excluded from the study, only the type iii degeneration, as per the modic classification, constituted grounds for exclusion), no pain or reduction of mobility in the lumbosacral section, other disorders of the spine (spondylolisthesis, fractures, tumors, rheumatic diseases, the cauda equina syndrome), pregnancy, defect symptoms, cardiovascular diseases, pacemakers, metal implants within the application area, sensory disorders, mental disorders, cancer, skin lesions within the area of application of the electrodes, psoriasis, scleroderma, and viral or bacterial infections. patients who had undergone spinal surgery, as well as those taking painkillers or anti - inflammatory drugs, were also excluded from the study. exclusion criteria referred also to patients with damage to the vestibule and/or a part of the vestibulocochlear nerve, with mnire syndrome, vestibular neuritis, sudden loss of function of the inner ear, as well as damage to the cerebellum, spinal cord and brainstem, resulting in balance disorders. a total of 127 patients were qualified for the therapy (ultimately, 124 patients completed the study) and assigned to 6 comparison groups (a flow diagram is presented in figure 1). group a consisted of 20 patients (all participants in this group completed the entire study program). treatment parameters were : alternating current, rectangular impulse, impulse duration 100 s, frequency of 100 hz, subjective dosage (until a distinctive sensation of the current flow was experienced, during the patient s habituation to the electrical stimulus, the therapist gradually increased the intensity during treatment to maintain the desired sensations), and 60-min duration of a single treatment. group b consisted of 20 patients (as in the previous group, all the patients completed the therapy). patients were treated with the transcutaneous electrical nerve stimulation (acupuncture - like tens). treatment parameters were : alternating current, rectangular impulse, impulse duration 200 s, frequency of 10 hz, subjective dosage (until distinctive muscle contraction, during habituation and decrease of the motion effect, the therapist gradually increased the intensity during treatment to maintain the desired muscle stimulation threshold), and 60-min duration of a single treatment. group c initially consisted of 22 patients, but 2 participants withdrew from further therapy due to viral infection and did not complete the treatment series (1 participant resigned after 4 sessions and the other after 6 sessions). one person had to discontinue the therapy after 3 sessions due to the occurrence of skin lesions within the area of application of the electrodes. in total, treatment parameters were : output voltage 100 v, alternating current, spike impulse, impulse duration 100 s, frequency of 100 hz, subjective dosage (until a distinctive sensation of the current flow was experienced, during the patient s habituation to the electrical stimulus, the therapist gradually increased intensity during treatment to maintain the desired sensations), and 50-min duration of a single treatment. group d initially consisted of 22 patients, but 1 person had to resign from further therapy due to aggravation of symptoms and started taking analgesics. subjects in group d were treated with electrotherapy using medium - frequency currents (interferential current stimulation). treatment parameters were : alternating current, sinusoidal impulse, impulse duration 100 s, basic frequency 4000 hz, alternating frequency 50100 hz, subjective dosage (until a distinctive sensation of the flowing current was experienced, during the patient s habituation to the electrical stimulus, the therapist gradually increased the intensity during treatment to maintain the desired sensations), and 20 min duration of a single treatment. treatment parameters were : pulsed current, sinusoidal impulse, impulse duration and frequency (sequentially df 10 ms, 100 hz, cp 10 ms, 50100 hz, lp 10 ms, 50100 hz, but with an alternating amplitude), subjective dosage (until a distinctive sensation of the flowing current is experienced, during the patient s habituation to the electrical stimulus the therapist gradually increased the intensity during treatment to maintain the desired sensations), and 9-min duration of a single treatment (df, lp, and cp were 3 min each). in all patients treated with physical therapy, the electrodes were placed in the lumbar region in the posterior axillary line (figures 2, 3). however, patients in group f (21 patients, control group) were treated only by means of motor improvement exercises. the stabilization training [57 ] included : myofascial release techniques for the erector spinae muscle, activation techniques for the neutral position of the lumbo - pelvic - hip complex and deep muscles, training the activation of proper breathing and the transversus abdominis muscle, exercising the coordination of superficial and deep trunk muscles, and postural and dynamic training. duration of a training session was 45 min (5 times a week, from monday to friday). in the comparison groups a, b, c, d, and e, patients treated with electrical therapy performed basic therapy exercises in accordance with the same methodology as patients in group f. patients in all the comparison groups (with the exception of group f, in which only the daily motor improvement exercises were used for 3 weeks) were subjected to a series of 15 treatments, 5 times a week (monday to friday) for a period of 3 weeks. the treatments were performed with the ionoson expert current generators (physio med electromedizin, germany), which were calibrated before treatment of each patient using the measurement system and the serial connection of the ionoson device to a cathodal oscilloscope and a decade resistor (electrical circuit loaded with the resistance of 10 k, as the average resistance of the human body) in order to verify the repeatability, durability, and stability of the treatment parameters generated by the electrostimulator. patients in all the comparison groups were homogeneous in terms of basic characteristics specific for the studied populations (table 1). the groups were also homogeneous as regards the initial measurements concerning pain assessment, functional state, mobility range, and body posture. in order to analyze the therapeutic progress for the subjective assessment of pain and functional capacity as well as the assessment of the degree of disability, the following tests were performed : the vas (visual analogue scale) pain assessment scale was used for subjective assessment of the experienced pain, in which the patient assesses the experienced pain on a simple scale from 0 to 10, where 0 denotes lack of pain and 10 denotes the strongest pain. the modified laitinen pain scale was used to assess 4 indicators : pain intensity, frequency of pain occurrence, use of analgesics, and limitations of mobility. the oswestry questionnaire (the oswestry low back pain disability questionnaire, oswestry disability index [odi ]) was used to evaluate the functional ability of patients ; it is a widely recognized and reliable scale for evaluation of patients with low back pain. when answering the individual questions, the patient can choose 1 of the 6 options scored from 0 to 5 : a 0 points ; b 1 point ; c 2 points ; d 3 points ; e 4 points ; f 5 points. after summing the scores for all questions, the oswestry disability index is as follows : no disability (04 points) ; minimal disability (514 points) ; moderate disability (1524 points) ; severe disability (2534 points) ; and full disability (3550 points). the roland - morris disability questionnaire (rm) was used to assess the degree of disability in patients with low back pain and reflects the condition of the patient on the day of the examination. each yes answer scores 1 point and each no answer scores 0 points. after summing the scores for all questions, the roland - morris disability index is as follows : no disability (03 points) ; minimal disability (410 points) ; moderate disability (1117 points) ; severe disability (1824 points). the lasgue test was used to measure the mobility range in the hip joint on the side of the herniated disc in the course of spinal discopathy. the examiner then slowly lifts one of the patient s legs while the knee is straight at the joint, until pain occurs. while the patient is in a standing position, the examiner marks 2 points on the patient s skin : at 10 cm above the line connecting the posterior superior iliac spines, and then at 5 cm below that line. the patient then slowly bends down as far as possible, while keeping the knees straight. the mobility range measurements were carried out by the same technician (each measurement was an arithmetic mean of 5 trials). for the purposes of this clinical study, a self - estimation of error of the person performing the measurements was calculated. for each of 15 randomly selected participants, 20 more measurements were taken using the lasgue test and the schober s test (600 measurements in total). the absolute measurement error (x) was calculated using the formula : then the relative error (x) was estimated, using the formula : the mean percentage error (relative error expressed in percentage points) was then calculated for all the 20 measurements for the lasgue and schober tests. the resulting measurement error, in accordance with the proprietary calculations, was as follows : the arithmetic mean of the measurement error was 5.88%, and the standard deviation was 3.73% for the lasgue test, and the mean was 3.45%, and the standard deviation was 1.04% for the schober test. the evaluation was performed using a double - plate stabilographic platform equipped with a computer - aided posturographic system, manufactured by cq elektronik system (poland), model cq stab2p. the measurement error was 0.86%. for each patient, 2 trials were carried out : the first trial with eyes open in full visual control, and the second trial with eyes closed, without visual control. the subjects were in a habitual, upright position, standing barefoot on the posturographic platform (feet apart in line with their hips, arms down along their bodies, head facing forward, with eyes fixed on a designated point placed at eye level about 1.5 m away) (figure 4). statistical analyses of the basic posturographic parameters were performed to compare balance conditions in the tested group of patients. the following parameters were analyzed : total path length [mm ], i.e. the total sway of the center of pressure of the subject s feet during the trial (30 s), in millimeters ; anterio / posterior path length [mm ] ; medio / lateral path length [mm ] ; mean amplitude (radius) [mm ] ; and mean anterio / posterior amplitude [mm ]. the above postural stability tests were carried out both before the therapy process and after its completion. the homogeneity of distribution of patients characteristics in all groups was analyzed with the chi - square test in the highest reliability version () and the kruskal - wallis homogeneity test. for dependent variables, the nonparametric wilcoxon s matched pairs test was used, and for independent variables we used the nonparametric kruskal - wallis variance analysis. the tukey post hoc multiple comparisons test was used to identify the exact dependencies resulting from the variance analysis between individual groups. the research project was approved by the bioethics commission of the academy of physical education in katowice (no. the study included patients with low back pain, referred for physical therapy to the clinical research lab of the public higher medical professional school in opole, poland. qualification of patients was made by a team composed of an orthopedist, a neurologist, a neurosurgeon, an internist, a radiologist, and a physiotherapist. patients qualified for the study suffered from the l5-s1 discopathy, chronic radiating pain, and pseudoradicular syndrome, and had no previous surgical procedures within the spinal area. the patients were at least 18 years old and had valid certificates of the mri examination, confirming the diagnosis of low back pain syndrome (at least type iii changes in accordance with the modic classification in the l5s1 spine section). on the other hand, the allocation of patients who positively underwent the qualification procedure to specific groups was random (based on a computer number generator). exclusion criteria referred to patients who were diagnosed with : acute pain (less than 6 months), the radicular syndrome, discopathy of other spine regions (patients with early type i and ii changes were not excluded from the study, only the type iii degeneration, as per the modic classification, constituted grounds for exclusion), no pain or reduction of mobility in the lumbosacral section, other disorders of the spine (spondylolisthesis, fractures, tumors, rheumatic diseases, the cauda equina syndrome), pregnancy, defect symptoms, cardiovascular diseases, pacemakers, metal implants within the application area, sensory disorders, mental disorders, cancer, skin lesions within the area of application of the electrodes, psoriasis, scleroderma, and viral or bacterial infections. patients who had undergone spinal surgery, as well as those taking painkillers or anti - inflammatory drugs, were also excluded from the study. exclusion criteria referred also to patients with damage to the vestibule and/or a part of the vestibulocochlear nerve, with mnire syndrome, vestibular neuritis, sudden loss of function of the inner ear, as well as damage to the cerebellum, spinal cord and brainstem, resulting in balance disorders. a total of 127 patients were qualified for the therapy (ultimately, 124 patients completed the study) and assigned to 6 comparison groups (a flow diagram is presented in figure 1). group a consisted of 20 patients (all participants in this group completed the entire study program). treatment parameters were : alternating current, rectangular impulse, impulse duration 100 s, frequency of 100 hz, subjective dosage (until a distinctive sensation of the current flow was experienced, during the patient s habituation to the electrical stimulus, the therapist gradually increased the intensity during treatment to maintain the desired sensations), and 60-min duration of a single treatment. group b consisted of 20 patients (as in the previous group, all the patients completed the therapy). patients were treated with the transcutaneous electrical nerve stimulation (acupuncture - like tens). treatment parameters were : alternating current, rectangular impulse, impulse duration 200 s, frequency of 10 hz, subjective dosage (until distinctive muscle contraction, during habituation and decrease of the motion effect, the therapist gradually increased the intensity during treatment to maintain the desired muscle stimulation threshold), and 60-min duration of a single treatment. group c initially consisted of 22 patients, but 2 participants withdrew from further therapy due to viral infection and did not complete the treatment series (1 participant resigned after 4 sessions and the other after 6 sessions). one person had to discontinue the therapy after 3 sessions due to the occurrence of skin lesions within the area of application of the electrodes. in total, treatment parameters were : output voltage 100 v, alternating current, spike impulse, impulse duration 100 s, frequency of 100 hz, subjective dosage (until a distinctive sensation of the current flow was experienced, during the patient s habituation to the electrical stimulus, the therapist gradually increased intensity during treatment to maintain the desired sensations), and 50-min duration of a single treatment. group d initially consisted of 22 patients, but 1 person had to resign from further therapy due to aggravation of symptoms and started taking analgesics. subjects in group d were treated with electrotherapy using medium - frequency currents (interferential current stimulation). treatment parameters were : alternating current, sinusoidal impulse, impulse duration 100 s, basic frequency 4000 hz, alternating frequency 50100 hz, subjective dosage (until a distinctive sensation of the flowing current was experienced, during the patient s habituation to the electrical stimulus, the therapist gradually increased the intensity during treatment to maintain the desired sensations), and 20 min duration of a single treatment. treatment parameters were : pulsed current, sinusoidal impulse, impulse duration and frequency (sequentially df 10 ms, 100 hz, cp 10 ms, 50100 hz, lp 10 ms, 50100 hz, but with an alternating amplitude), subjective dosage (until a distinctive sensation of the flowing current is experienced, during the patient s habituation to the electrical stimulus the therapist gradually increased the intensity during treatment to maintain the desired sensations), and 9-min duration of a single treatment (df, lp, and cp were 3 min each). in all patients treated with physical therapy, the electrodes were placed in the lumbar region in the posterior axillary line (figures 2, 3). however, patients in group f (21 patients, control group) were treated only by means of motor improvement exercises. the stabilization training [57 ] included : myofascial release techniques for the erector spinae muscle, activation techniques for the neutral position of the lumbo - pelvic - hip complex and deep muscles, training the activation of proper breathing and the transversus abdominis muscle, exercising the coordination of superficial and deep trunk muscles, and postural and dynamic training. duration of a training session was 45 min (5 times a week, from monday to friday). in the comparison groups a, b, c, d, and e, patients treated with electrical therapy performed basic therapy exercises in accordance with the same methodology as patients in group f. patients in all the comparison groups (with the exception of group f, in which only the daily motor improvement exercises were used for 3 weeks) were subjected to a series of 15 treatments, 5 times a week (monday to friday) for a period of 3 weeks. the treatments were performed with the ionoson expert current generators (physio med electromedizin, germany), which were calibrated before treatment of each patient using the measurement system and the serial connection of the ionoson device to a cathodal oscilloscope and a decade resistor (electrical circuit loaded with the resistance of 10 k, as the average resistance of the human body) in order to verify the repeatability, durability, and stability of the treatment parameters generated by the electrostimulator. patients in all the comparison groups were homogeneous in terms of basic characteristics specific for the studied populations (table 1). the groups were also homogeneous as regards the initial measurements concerning pain assessment, functional state, mobility range, and body posture. in order to analyze the therapeutic progress for the subjective assessment of pain and functional capacity as well as the assessment of the degree of disability, the following tests were performed : the vas (visual analogue scale) pain assessment scale was used for subjective assessment of the experienced pain, in which the patient assesses the experienced pain on a simple scale from 0 to 10, where 0 denotes lack of pain and 10 denotes the strongest pain. the modified laitinen pain scale was used to assess 4 indicators : pain intensity, frequency of pain occurrence, use of analgesics, and limitations of mobility. the oswestry questionnaire (the oswestry low back pain disability questionnaire, oswestry disability index [odi ]) was used to evaluate the functional ability of patients ; it is a widely recognized and reliable scale for evaluation of patients with low back pain. when answering the individual questions, the patient can choose 1 of the 6 options scored from 0 to 5 : a 0 points ; b 1 point ; c 2 points ; d 3 points ; e 4 points ; f 5 points. after summing the scores for all questions, the oswestry disability index is as follows : no disability (04 points) ; minimal disability (514 points) ; moderate disability (1524 points) ; severe disability (2534 points) ; and full disability (3550 points). the roland - morris disability questionnaire (rm) was used to assess the degree of disability in patients with low back pain and reflects the condition of the patient on the day of the examination. each yes answer scores 1 point and each no answer scores 0 points. after summing the scores for all questions, the roland - morris disability index is as follows : no disability (03 points) ; minimal disability (410 points) ; moderate disability (1117 points) ; severe disability (1824 points). the lasgue test was used to measure the mobility range in the hip joint on the side of the herniated disc in the course of spinal discopathy. the examiner then slowly lifts one of the patient s legs while the knee is straight at the joint, until pain occurs. while the patient is in a standing position, the examiner marks 2 points on the patient s skin : at 10 cm above the line connecting the posterior superior iliac spines, and then at 5 cm below that line. the patient then slowly bends down as far as possible, while keeping the knees straight. the mobility range measurements were carried out by the same technician (each measurement was an arithmetic mean of 5 trials). for the purposes of this clinical study, a self - estimation of error of the person performing the measurements was calculated. for each of 15 randomly selected participants, 20 more measurements were taken using the lasgue test and the schober s test (600 measurements in total). the absolute measurement error (x) was calculated using the formula : then the relative error (x) was estimated, using the formula : the mean percentage error (relative error expressed in percentage points) was then calculated for all the 20 measurements for the lasgue and schober tests. the resulting measurement error, in accordance with the proprietary calculations, was as follows : the arithmetic mean of the measurement error was 5.88%, and the standard deviation was 3.73% for the lasgue test, and the mean was 3.45%, and the standard deviation was 1.04% for the schober test. the evaluation was performed using a double - plate stabilographic platform equipped with a computer - aided posturographic system, manufactured by cq elektronik system (poland), model cq stab2p. 2 trials were carried out : the first trial with eyes open in full visual control, and the second trial with eyes closed, without visual control. the subjects were in a habitual, upright position, standing barefoot on the posturographic platform (feet apart in line with their hips, arms down along their bodies, head facing forward, with eyes fixed on a designated point placed at eye level about 1.5 m away) (figure 4). statistical analyses of the basic posturographic parameters were performed to compare balance conditions in the tested group of patients. the following parameters were analyzed : total path length [mm ], i.e. the total sway of the center of pressure of the subject s feet during the trial (30 s), in millimeters ; anterio / posterior path length [mm ] ; medio / lateral path length [mm ] ; mean amplitude (radius) [mm ] ; and mean anterio / posterior amplitude [mm ]. the above postural stability tests were carried out both before the therapy process and after its completion. the studied parameters were analyzed using the statistica statistical software ver. 10.0 (statsoft, dell inc. the homogeneity of distribution of patients characteristics in all groups was analyzed with the chi - square test in the highest reliability version () and the kruskal - wallis homogeneity test. for dependent variables, the nonparametric wilcoxon s matched pairs test was used, and for independent variables we used the nonparametric kruskal - wallis variance analysis. the tukey post hoc multiple comparisons test was used to identify the exact dependencies resulting from the variance analysis between individual groups. after completion of the therapy, all the comparison groups demonstrated a statistically significant reduction of pain as compared to the initial values, measured using the vas scale (table 2). similarly, a subjective reduction of pain was recorded using the laitinen questionnaire (table 3). however, the intergroup analysis demonstrated that the highest analgesic effect was recorded in group d (interferential current stimulation), which proved to be a significantly better result than in groups a (conventional tens), b (acupuncture - like tens), and c (high - voltage electrical stimulation [hves ]). no statistically significant differences were observed between groups a, b, and c. the lowest analgesic effect was observed in group e (diadynamic currents [dd ]) and f (control group), at a similar level in both groups. the above was confirmed both by the results of measurements performed using the vas scale (figure 5) and the laitinen questionnaire (figure 6). after 3 weeks of treatment, all the comparison groups demonstrated a statistically significant improvement of functional ability, measured using the oswestry questionnaire (table 4). similarly, a subjective improvement of patients ability was recorded using the roland - morris questionnaire (table 5). however, on the basis of an intergroup analysis, we found that the highest percentual improvement of the patients functional ability measured with the oswestry questionnaire took place in group d (interferential current stimulation), giving a significantly more beneficial effect than in groups a (conventional tens), b (acupuncture - like tens), and c (hves). no statistically significant differences were observed between groups a, b, and c. the lowest effect was observed in group e (dd) and f (control group), at a similar level in both groups (figure 7). in case of intergroup comparisons regarding the subjective experience of ability measured with the roland - morris questionnaire, the greatest progress was observed in group d (interferential current stimulation). in the remaining groups, immediately after completion of therapy, the comparison groups demonstrated a statistically significant increase of mobility in the hip joint as compared to the pre - therapy state, measured using the lasgue test (table 6). an improved mobility in the low back region was also recorded in the schober s test (table 7). on the basis of an intergroup analysis, we found that the highest percentual improvement in the lasgue test took place in group d (interferential current stimulation), giving a significantly better result than in groups a (conventional tens), b (acupuncture - like tens), and c (hves). no statistically significant differences were observed between groups a, b, and c. the least progress was recorded in group e (dd) and f (control group), at a similar level in both groups (figure 9). the comparison of intergroup results of the schober s test indicated that groups a (conventional tens), b (pseudo - acupuncture tens), c (hves), and d (interference currents) gained a significant advantage over groups e (dd) and f (control group) (figure 10). after completion of therapy, tests on the stabilometric platform clearly demonstrated a statistically significant improvement in body posture as regards the total path length of sway of the center of pressure during a time trial (table 8) and the path length in the anterio - posterior sway (table 9) in comparison to the initial state. these parameters were significantly reduced in all the comparison groups (in particular with eyes closed), which confirms that patients with low back pain had a more stable body posture after the therapy. in case of measurement of the path in the lateral sway, a beneficial reduction was also recorded in the studied groups, although these changes were not statistically significant (table 10). interesting changes occurred in measurements of the mean radius of the center of pressure sway, as the beneficial reduction of this parameter and improvement of postural stability took place in the closed - eyes trial (table 11). in trials under visual control, the radius was also reduced in all groups, but this change was significantly smaller. the analysis of variance showed that the greatest percentual reduction in the length of the total path of the center of pressure sway in a time trial occurred in group d (interference currents). these parameters were also slightly reduced in the remaining groups, especially group a (conventional tens), b (acupuncture - like tens), and c (hves), but despite this trend, no statistically significant difference was obtained in relation to groups e (dd) and f (control group). a similar situation was observed in the case of closed - eyes trial, where group d had an even more significant advantage over the other groups. the analysis of percentual shortening of the path length in the anterio - posterior sway of the patients center of pressure indicates that the outcome of this process was the most beneficial in group d (interferential current stimulation). in the remaining groups, the phenomenon occurred with a slightly lower intensity. on the other hand, in the percentage analysis of reduction of the mean radius of center of pressure sway in trials under visual control, there were no significant changes, and the recorded changes occurred at a similar level in all groups. interestingly, in the eyes - closed trial, patients in group d (interferential current stimulation) had better scores than those obtained in other groups. however, no statistically significant differences were detected, although an evident trend was observed regarding changes in benefit of group d in relation to the other groups. observation of the percentual reduction of the mean lateral sway of the center of pressure in the eyes - open trial did not show a visible improvement of this parameter in the studied groups (reduction of sway was small and occurred at a similar level in all patients irrespectively of the treatment method). no statistically significant intergroup differences were noted. all the more interesting was the significant advantage of group d (interferential current stimulation) in relation to the other groups during the trial without visual control. in group d, a statistically significant difference was noted in relation to other groups. after completion of the therapy, all the comparison groups demonstrated a statistically significant reduction of pain as compared to the initial values, measured using the vas scale (table 2). similarly, a subjective reduction of pain was recorded using the laitinen questionnaire (table 3). however, the intergroup analysis demonstrated that the highest analgesic effect was recorded in group d (interferential current stimulation), which proved to be a significantly better result than in groups a (conventional tens), b (acupuncture - like tens), and c (high - voltage electrical stimulation [hves ]). no statistically significant differences were observed between groups a, b, and c. the lowest analgesic effect was observed in group e (diadynamic currents [dd ]) and f (control group), at a similar level in both groups. the above was confirmed both by the results of measurements performed using the vas scale (figure 5) and the laitinen questionnaire (figure 6). after 3 weeks of treatment, all the comparison groups demonstrated a statistically significant improvement of functional ability, measured using the oswestry questionnaire (table 4). similarly, a subjective improvement of patients ability was recorded using the roland - morris questionnaire (table 5). however, on the basis of an intergroup analysis, we found that the highest percentual improvement of the patients functional ability measured with the oswestry questionnaire took place in group d (interferential current stimulation), giving a significantly more beneficial effect than in groups a (conventional tens), b (acupuncture - like tens), and c (hves). no statistically significant differences were observed between groups a, b, and c. the lowest effect was observed in group e (dd) and f (control group), at a similar level in both groups (figure 7). in case of intergroup comparisons regarding the subjective experience of ability measured with the roland - morris questionnaire, the greatest progress was observed in group d (interferential current stimulation). in the remaining groups, immediately after completion of therapy, the comparison groups demonstrated a statistically significant increase of mobility in the hip joint as compared to the pre - therapy state, measured using the lasgue test (table 6). an improved mobility in the low back region was also recorded in the schober s test (table 7). on the basis of an intergroup analysis, we found that the highest percentual improvement in the lasgue test took place in group d (interferential current stimulation), giving a significantly better result than in groups a (conventional tens), b (acupuncture - like tens), and c (hves). no statistically significant differences were observed between groups a, b, and c. the least progress was recorded in group e (dd) and f (control group), at a similar level in both groups (figure 9). the comparison of intergroup results of the schober s test indicated that groups a (conventional tens), b (pseudo - acupuncture tens), c (hves), and d (interference currents) gained a significant advantage over groups e (dd) and f (control group) (figure 10). after completion of therapy, tests on the stabilometric platform clearly demonstrated a statistically significant improvement in body posture as regards the total path length of sway of the center of pressure during a time trial (table 8) and the path length in the anterio - posterior sway (table 9) in comparison to the initial state. these parameters were significantly reduced in all the comparison groups (in particular with eyes closed), which confirms that patients with low back pain had a more stable body posture after the therapy. in case of measurement of the path in the lateral sway, a beneficial reduction was also recorded in the studied groups, although these changes were not statistically significant (table 10). interesting changes occurred in measurements of the mean radius of the center of pressure sway, as the beneficial reduction of this parameter and improvement of postural stability took place in the closed - eyes trial (table 11). in trials under visual control, the radius was also reduced in all groups, but this change was significantly smaller. the analysis of variance showed that the greatest percentual reduction in the length of the total path of the center of pressure sway in a time trial occurred in group d (interference currents). these parameters were also slightly reduced in the remaining groups, especially group a (conventional tens), b (acupuncture - like tens), and c (hves), but despite this trend, no statistically significant difference was obtained in relation to groups e (dd) and f (control group). a similar situation was observed in the case of closed - eyes trial, where group d had an even more significant advantage over the other groups. the analysis of percentual shortening of the path length in the anterio - posterior sway of the patients center of pressure indicates that the outcome of this process was the most beneficial in group d (interferential current stimulation). in the remaining groups, the phenomenon occurred with a slightly lower intensity. on the other hand, in the percentage analysis of reduction of the mean radius of center of pressure sway in trials under visual control, there were no significant changes, and the recorded changes occurred at a similar level in all groups. interestingly, in the eyes - closed trial, patients in group d (interferential current stimulation) had better scores than those obtained in other groups. however, no statistically significant differences were detected, although an evident trend was observed regarding changes in benefit of group d in relation to the other groups. observation of the percentual reduction of the mean lateral sway of the center of pressure in the eyes - open trial did not show a visible improvement of this parameter in the studied groups (reduction of sway was small and occurred at a similar level in all patients irrespectively of the treatment method). all the more interesting was the significant advantage of group d (interferential current stimulation) in relation to the other groups during the trial without visual control. in group d this condition may also cause pain radiating along the lower limbs, muscle weakness due to compression and irritation of nerve roots, as well as limitation of spinal mobility. these symptoms may appear at any stage of the disease, disturbing the function of the locomotor system. they are also a cause of disability and related social factors, which entails increased expenditure on, among others, diagnostics and treatment. therefore, it is necessary to search for effective treatment methods for back pain syndromes. one of the treatment methods is physical therapy, which helps to reduce the disease symptoms. although the available literature contains a large number of proposals for conservative treatment of low back pain using electrical therapy, such publications usually concern only 1 type (or, in some cases, 2 types in a direct comparison under 1 clinical study) of electric current. such articles contain a number of limitations and weaknesses, which makes it difficult to carry out an unequivocal clinical assessment. studies conducted by brazilian researchers confirm the high effectiveness of interference currents and transcutaneous electrical nerve stimulation (tens) in low back pain therapy. compared the effects of the tens treatment and interferential current stimulation (ifc) treatment in patients with nonspecific chronic low back pain. the study involved a group of 150 patients, randomly assigned to 3 comparison groups. the first group consisted of patients who were treated with tens therapy, the second group was treated with the ifc currents, and the third group was not treated with any physical stimulus. patients in the first and second group underwent treatment for 30 min during 10 consecutive days. in all patients, pain level was assessed using the visual analogue pain scale (vas) and the mcgill pain questionnaire. results of the study demonstrate that the tens and ifc therapy brought about significant effects as in reduction of pain intensity, improvement of disability, and reduction in the amount of non - steroidal anti - inflammatory drugs used, in comparison to the control group. however, no significant differences were noted between patients from the first and the second group. other brazilian researchers evaluated the effectiveness of the interferential current stimulation (ifc) therapy. correa. conducted a randomized clinical trial involving 150 patients with a chronic, nonspecific low back pain syndrome, which confirmed the beneficial therapeutic effect of the ifc.. demonstrated in their publication that the effectiveness of interferential currents was higher than that of superficial massage applied to the lumbar region of the spine. patients from the first group were subjected to a series of treatments using ifc, whereas patients in the second group underwent a series of massage treatments. in both groups, treatments were performed for 10 weeks (a total of 20 treatments, 2 times a week). subjective pain assessment was performed using the visual analogue pain scale (vas), while the oswestry and the roland - morris questionnaires were used to evaluate the functional ability of patients. after the completed therapy, it was found that in comparison to superficial massage, the interference current led to significantly more improvement of disability, reduction of pain, and increase of quality of life. turkish researchers demonstrated that tens and exercise increased quality of life and reduced pain in patients with low back pain. in the first group patients performed aerobic and general development exercises, the second group performed general development exercises and was treated with physical therapy (conventional tens), and in the third group the patients performed only general development exercises. before the therapy and after its completion, spine mobility was assessed with schober s test, pain intensity was assessed with the visual analogue pain scale (vas), and the disability assessment was performed using the roland - morris questionnaire. an assessment was also made of the general mental condition of patients, using the beck depression inventory (bdi). on the basis of the obtained effects, only the second study group demonstrated a statistically significant improvement as regards the measured parameters and the experienced pain. however, the literature also provides examples of cases where electrotherapy had low effectiveness or only a short - term analgesic effect. mcloughlin. indicated a lack of analgesic effect after the application of high - voltage electrical stimulation. ambiguous results of a study in which tens was used in therapy of low back pain was presented in a publication by buchmuller.. the study involved 236 persons suffering from low back pain, from 21 treatment centers in france. the first group was made up of 117 patients who were treated with active tens treatments, while the second group, consisting of 119 patients, was subjected to simulated tens treatments. the functional state of patients was assessed using the roland - morris questionnaire, and pain intensity was measured in accordance with the visual analogue pain scale (vas). surveys were conducted before the commencement of the therapy, after 2 and 6 weeks, and at 3 months after completion of the study. independently of the study report, patients kept a diary to record their assessment of the intensity of pain. analysis of the obtained results demonstrated similar reactions in the group of patients who underwent the active tens treatments and in the placebo group as regards the assessment of functional state and in relation to the vas scale. however, the assessments of pain intensity recorded in patients diaries demonstrated a highly significant difference in favor of the active tens treatments, which shows a strong placebo effect of this method. in the meta - analysis presented by khadilkar., whose aim was to determine the effectiveness of tens in the therapy of chronic pain of the lumbar section of the spine, a series of equivocal data was recorded, confirming the use of the tens as an analgesic. the authors, while performing an in - depth assessment, noted inter alia the non - uniform methodology, the discrepancies in inclusion and exclusion criteria, and the differing therapy durations. it would be valuable to have results from a large, multi - center research project using control groups (quasi - electrotherapy) and thorough randomization, as well as to unify the methodology of the performed treatments. particular attention should be given to the long - term benefits which may possibly be observed after the application of tens. the novelty of our study consists in an unambiguous assessment of popular methods of electrical therapy used in low back pain treatment under a single, prospective, randomized clinical pilot trial, based on strict inclusion and exclusion criteria, using both modern objective measurements and subjective measurements, with a uniform statistical analysis and a comprehensive and multi - faceted observation of the achieved results. to the best of our knowledge, the present study is the first wide - ranging scientific study of low back pain electrotherapy in accordance with the guidelines of the evidence - based physiotherapy. none of the current publications made such a detailed diagnostics or a classification based on the radiological modic assessment criteria. this allowed for a very representative population, which was recruited for the purpose of this project. in addition, other researchers did not use calibration of the electrostimulator before each treatment. irrespective of the innovative character of our pilot research and the elements of novelty, this work also contains a number of limitations. it would certainly be worthwhile to supplement the project in the future with other modern and objective measurement tools, such as muscle electromyography, the biodex system movement analysis, and tensiometry. also, the study did not involve blind trials or placebo effect assessment. in 1 of the comparison groups, patients underwent only the standard functional training and were not treated with any electrotherapeutical treatment whatsoever, which constituted a point of reference in relation to the exposed groups and which is permitted in the methodology of medical research publications. however, using the simulated treatments and the so - called single - blind trial would certainly constitute an interesting addition and would also significantly raise the profile of this study. a weakness of this research was also the relatively high measurement error (although in most cases the researchers do not even perform this kind of analysis or self - reflection) during the observation of mobility of the hip joint and the lumbar region of the spine. we endeavored to mitigate it by making our own error estimations and by making sure that all measurements were made by the same person (an average of the 5 performed trials)., it will also be necessary to perform an assessment of remote results, which will allow us to estimate the sustainability of remission achieved due to electrical therapy. the novelty of our study consists in an unambiguous assessment of popular methods of electrical therapy used in low back pain treatment under a single, prospective, randomized clinical pilot trial, based on strict inclusion and exclusion criteria, using both modern objective measurements and subjective measurements, with a uniform statistical analysis and a comprehensive and multi - faceted observation of the achieved results. to the best of our knowledge, the present study is the first wide - ranging scientific study of low back pain electrotherapy in accordance with the guidelines of the evidence - based physiotherapy. none of the current publications made such a detailed diagnostics or a classification based on the radiological modic assessment criteria. this allowed for a very representative population, which was recruited for the purpose of this project. in addition, other researchers did not use calibration of the electrostimulator before each treatment. irrespective of the innovative character of our pilot research and the elements of novelty, this work also contains a number of limitations. it would certainly be worthwhile to supplement the project in the future with other modern and objective measurement tools, such as muscle electromyography, the biodex system movement analysis, and tensiometry. also, the study did not involve blind trials or placebo effect assessment. in 1 of the comparison groups, patients underwent only the standard functional training and were not treated with any electrotherapeutical treatment whatsoever, which constituted a point of reference in relation to the exposed groups and which is permitted in the methodology of medical research publications. however, using the simulated treatments and the so - called single - blind trial would certainly constitute an interesting addition and would also significantly raise the profile of this study. a weakness of this research was also the relatively high measurement error (although in most cases the researchers do not even perform this kind of analysis or self - reflection) during the observation of mobility of the hip joint and the lumbar region of the spine. we endeavored to mitigate it by making our own error estimations and by making sure that all measurements were made by the same person (an average of the 5 performed trials)., it will also be necessary to perform an assessment of remote results, which will allow us to estimate the sustainability of remission achieved due to electrical therapy. our conducted research indicates that using electrostimulation with interferential current stimulation penetrating deeper into the tissues results in a significant and long - term elimination of pain, and an improvement of functional ability of patients suffering from low back pain on the basis of an analysis of both subjective and objective parameters. although tens currents and hves are helpful in treatment of discopathy of the lower region of the spine, use of interference currents led to greater remission of symptoms. on the other hand, the research indicates that the use of diadynamic currents appears to be useless in the course of degenerative proliferative disease of the spine (within the scope studied in this paper). | backgroundin the currently available research publications on electrical therapy of low back pain, generally no control groups or detailed randomization were used, and such studies were often conducted with relatively small groups of patients, based solely on subjective questionnaires and pain assessment scales (lacking measurement methods to objectify the therapeutic progress). the available literature also lacks a comprehensive and large - scale clinical study. the purpose of this study was to assess the effects of treating low back pain using selected electrotherapy methods. the study assesses the influence of individual electrotherapeutic treatments on reduction of pain, improvement of the range of movement in lower section of the spine, and improvement of motor functions and mobility.material/methodsthe 127 patients qualified for the therapy (ultimately, 123 patients completed the study) and assigned to 6 comparison groups : a conventional tens, b acupuncture - like tens, c high - voltage electrical stimulation, d interferential current stimulation, e diadynamic current, and f control group.resultsthe research showed that using electrical stimulation with interferential current penetrating deeper into the tissues results in a significant and more efficient elimination of pain, and an improvement of functional ability of patients suffering from low back pain on the basis of an analysis of both subjective and objective parameters. the tens currents and high voltage were helpful, but not as effective. the use of diadynamic currents appears to be useless.conclusionsselected electrical therapies (interferential current, tens, and high voltage) appear to be effective in treating chronic low back pain. |
reproductive efficiency is an important strategy for maximizing the productivity of cattle herds. among the current strategies used to improve reproductive performance, ovulation synchronization has become the best technique for optimizing herd management and providing genetic improvements with greater efficiency. among beef cattle, approximately 40 to 60% of inseminated females become pregnant after the first cycle of fixed - time artificial insemination (ftai), with slight variations in rates depending on several factors including the animals, farm management, body condition score, postpartum time and hormonal treatments. therefore, females that do not conceive after the first service need to be re - inseminated as soon as possible to increase the reproductive performance of the herd. if these females are not re - inseminated in a short period of time, the interval between calving and conception increases, decreasing the reproductive efficiency of the herd. the resynchronization of females who failed the first ftai is an effective strategy to increase the number of pregnancies achieved by ai during a short breeding season. ovulation resynchronization can increase the percentage of non - pregnant cows that are subjected to a second insemination and be an advantageous strategy when the number of bulls is insufficient to mate with all of the cows that did not become pregnant after the first ftai. during resynchronization, all cows inseminated in the first round of ftai should be evaluated by ultrasonography, and those that did not become pregnant receive the same hormonal protocol for a second round of ftai. one of the most common hormonal protocols for ftai or resynchronization in cattle involves the use of a progesterone source, such as an intravaginal device or norgestomet ear implant, in combination with 1 mg of estradiol benzoate (eb), 0.5 mg of estradiol cypionate (ec) or 100 g of gnrh. temporary calf removal (tcr) for 48 to 54 h or 300 iu of equine chorionic gonadotropin (ecg) have also been successfully applied at the time of device withdrawal and in conjunction with a luteolytic agent and inducer of ovulation to improve the reproductive performance of resynchronized bos indicus cows. resynchronization protocols have been evaluated in different categories of female beef and dairy cattle (bos taurus), non - lactating cows and beef heifers (bos taurus), multiparous lactating beef cows (bos indicus), non - lactating cows and cycling beef heifers (bos indicus) and crossbred cows from bos indicus and bos taurus parents. nevertheless, no studies have examined different cohorts in a single resynchronization study, particularly with heifers or newly calved cows. therefore, the present study was conducted to investigate the conception rate in heifers, primiparous and multiparous lactating cows after a large - scale ftai program, followed by resynchronization using an estradiol / p4-based protocol. this study was conducted during the 2011/2012 breeding season in south america at latitude 2115'23 '' and longitude 522'29 ''. the climate in this region is tropical, with an average temperature of 24 and a rainy season from november to january. data for this study were collected from bos indicus (nelore) herds including heifers, as well as primiparous and multiparas lactating cows (n = 2,464) that were selected to receive a conventional protocol of ftai in two commercial beef farms in southern brazil under the same management. heifers (24 to 27 months old) and cows (36 to 180 months old) were selected based on an adequate body condition score (bcs) and postpartum period, normal estrous cycles and health status. the primiparous and multiparous lactating cows were 30 to 50 days postpartum (average 45 days), and only females with a bcs between 2.0 and 4.0 (average 3) on a scale of 1 to 5 were used for the ftai program. the herds were kept in an extensive system that allowed for continuous grazing of brachiaria spp. and were given ad libitum access to mineralized salt and water. on a random day of the estrous cycle (day 0), heifers (n = 903), primiparous lactating cows (n = 338) and multiparous lactating cows (n = 1,223) underwent a conventional estradiol / p4-based ftai protocol. the protocol consisted of the insertion of an intravaginal device containing 1 g of progesterone (p4) (dib ; msd animal health, brazil ; cows) or an ear implant containing 3 mg of norgestomet (crestar ; msd animal health ; heifers) and intramuscular (i.m.) administration of 2 mg of eb (gonadiol ; msd animal health). on day 8, the devices were removed and the animals received i.m. injections containing 250 g of cloprostenol (ciosin ; msd animal health), 300 iu of ecg (novormon ; msd animal health) and ec (1.0 mg for lactating cows and 0.5 mg for heifers, ecp ; zoetis, brazil). all females were inseminated by two experienced inseminators using frozen - thawed semen of bulls 48 h after device removal (fig. thirty days after the first ftai (day 28 to 32), all animals were subjected to a pregnancy diagnosis by transrectal ultrasonography (aloka ssd 500 ; aloka, japan) and non - pregnant females received the same hormonal treatment described above. heifers received norgestomet due to poor estrus synchronization results when a new p4-releasing intravaginal device (first use) was used. to resynchronize ovulation, all females were implanted with a previously used p4 source (device or implant) for 8 days. after their initial use, the devices / implants were individually washed with water, then soaked in a solution of ascorbic acid, citric acid and lactic acid (kilol - l ; quinabra, brazil) for approximately 10 minutes. the first evaluation occurred on day 30 (28 to 32 days), while the second was conducted 30 days later. in both evaluations, females were scanned by transrectal ultrasonography using an aloka ssd-500 ultrasound equipped with a 5 mhz linear transducer (aloka). the conception rate was calculated by dividing the number of pregnant cows by the total number of females inseminated on day 30 and 30 days after the second ftai. the conception rates corresponding to the first ftai, second ftai and overall rate within 30 days of the second ftai (first ftai + resynchronization) were compared across categories (heifers, primiparous cows and multiparous cows). a chi - square test was used to determine differences in conception rates among the categories of females. this study was conducted during the 2011/2012 breeding season in south america at latitude 2115'23 '' and longitude 522'29 ''. the climate in this region is tropical, with an average temperature of 24 and a rainy season from november to january. data for this study were collected from bos indicus (nelore) herds including heifers, as well as primiparous and multiparas lactating cows (n = 2,464) that were selected to receive a conventional protocol of ftai in two commercial beef farms in southern brazil under the same management. heifers (24 to 27 months old) and cows (36 to 180 months old) were selected based on an adequate body condition score (bcs) and postpartum period, normal estrous cycles and health status. the primiparous and multiparous lactating cows were 30 to 50 days postpartum (average 45 days), and only females with a bcs between 2.0 and 4.0 (average 3) on a scale of 1 to 5 were used for the ftai program. the herds were kept in an extensive system that allowed for continuous grazing of brachiaria spp. and were given ad libitum access to mineralized salt and water. on a random day of the estrous cycle (day 0), heifers (n = 903), primiparous lactating cows (n = 338) and multiparous lactating cows (n = 1,223) underwent a conventional estradiol / p4-based ftai protocol. the protocol consisted of the insertion of an intravaginal device containing 1 g of progesterone (p4) (dib ; msd animal health, brazil ; cows) or an ear implant containing 3 mg of norgestomet (crestar ; msd animal health ; heifers) and intramuscular (i.m.) administration of 2 mg of eb (gonadiol ; msd animal health). on day 8 injections containing 250 g of cloprostenol (ciosin ; msd animal health), 300 iu of ecg (novormon ; msd animal health) and ec (1.0 mg for lactating cows and 0.5 mg for heifers, ecp ; zoetis, brazil). all females were inseminated by two experienced inseminators using frozen - thawed semen of bulls 48 h after device removal (fig. thirty days after the first ftai (day 28 to 32), all animals were subjected to a pregnancy diagnosis by transrectal ultrasonography (aloka ssd 500 ; aloka, japan) and non - pregnant females received the same hormonal treatment described above. heifers received norgestomet due to poor estrus synchronization results when a new p4-releasing intravaginal device (first use) was used. to resynchronize ovulation, all females were implanted with a previously used p4 source (device or implant) for 8 days. after their initial use, the devices / implants were individually washed with water, then soaked in a solution of ascorbic acid, citric acid and lactic acid (kilol - l ; quinabra, brazil) for approximately 10 minutes. the first evaluation occurred on day 30 (28 to 32 days), while the second was conducted 30 days later. in both evaluations, females were scanned by transrectal ultrasonography using an aloka ssd-500 ultrasound equipped with a 5 mhz linear transducer (aloka). the conception rate was calculated by dividing the number of pregnant cows by the total number of females inseminated on day 30 and 30 days after the second ftai. the data are presented as proportions. the conception rates corresponding to the first ftai, second ftai and overall rate within 30 days of the second ftai (first ftai + resynchronization) were compared across categories (heifers, primiparous cows and multiparous cows). a chi - square test was used to determine differences in conception rates among the categories of females. of the 2,464 female bovines subjected to an estradiol / p4-based ftai protocol followed by ovulation resynchronization, 1,985 animals became pregnant, resulting in an average conception rate of 81% 30 days after the second ftai (table 1). heifers exhibited a higher conception rate (85% ; 770/903) than the primiparous cows (76% ; 257/338) or multiparous cows (78% ; 958/1,223 ; p = 0.0001). the average conception rate after the first ftai was 55% (1,367/2,464), and there were no differences between the conception rates of the heifers (57% ; 514/903), primiparous cows (51% ; 173/338) or multiparous cows (56% ; 680/1,223 ; p = 0.193). the average conception rate after the second ftai (resynchronization) was 56% (618/1,097), and the conception rate was higher in heifers (66% ; 256/389) than in the primiparous cows (51% ; 84/165) and multiparous cows (51% ; 278/543 ; p = 0.0001). data from the current study demonstrated that it is possible to achieve high pregnancy rates in a short period of the breeding season using only ftai after resynchronization of heifers, primiparous and multiparous lactating cows. the higher pregnancy rate after the second ftai in heifers should be considered strategic information for large scale ai programs in beef herds. thirty days after the first ftai procedure, we used 1 mg of eb in combination with a p4 source (intravaginal device or norgestomet ear implant) to induce a new follicular wave. a recent study evaluated the use of resynchronization employing a progestin - based ftai protocol in beef cattle (bos indicus) with two different inducers of new follicular waves (eb vs. gnrh). this study showed that both inducers resulted in similar pregnancy rates (70.5% and 63.2%, respectively). additionally, 1 mg of eb 22 days after ftai did not affect pre - established pregnancy. another study of previously inseminated lactating beef cows (bos taurus) used resynchronization with p4 + 1 mg of eb or p4 + 0.5 mg of ec. the p4 sources in combination with estrogen treatment did not decrease the conception rates, resulting in pregnancy rates of 86% and 65%, respectively. in the present study, the conception rate following the first ftai protocol did not differ among the cohorts evaluated (heifers (57%), primiparous cows (51%) and multiparous cows (56%)). additionally, these results were very similar to those observed by campos., who achieved conception rates ranging from 47 to 54% in nelore multiparous cows after evaluating data from the first ftai in an estrus resynchronization program. the second ftai resulted in higher conception rates in heifers than cows (66% vs. 51%, respectively), demonstrating that the category influences the conception rate of re - inseminated females. some field studies have shown that bos indicus cows exhibit different pregnancy rates following the first and second ftai depending on the category of the resynchronized females. in our field experience, there is a decrease in pregnancy rate following the second ftai relative to the rate after the first ftai of primiparous cows. in the present study the pregnancy rate was similar when a second synchronization of ovulation was performed. the resynchronized females from the present study achieved an average pregnancy rate of approximately 80% (after both the first and second ftai) in only 80 days of the breeding season. despite the better efficiency of resynchronization in heifers (85%), this protocol also provided promising results in primiparous (76%) and multiparous (78%) cows. by resynchronizing cycling heifers and non - lactating cows (bos indicus and crossbred bos indicus bos taurus), s filho. achieved a 75% pregnancy rate, which was similar to the data described here. similarly, campos. reported pregnancy rates of 76.6% and 74% following the resynchronization of lactating cows treated with 300 ui of ecg or temporary calf removal, respectively. lactating cows in the postpartum period represent a category of females with higher energy requirements. during this period, the cow 's nutritional status and presence of the calf exert strong influences on the female 's energetic balance that could negatively influence breeding potential and the success of ftai. remarkably, in the present study, primiparous cows achieved a conception rate similar to multiparous cows that was similar to the average found in other studies of multiparous cows (74 - 76%). in conclusion, ovulation resynchronization of females who failed the first ftai is a feasible and effective technique, providing an average conception rate of 80% in beef cattle. the best result was found in heifers, with a 66% pregnancy rate being observed after the second ftai and an 85% rate after the two ftai procedures. | this study was conducted to evaluate the influence of category (heifers, primiparous or multiparous cows) on pregnancy rates in a large scale resynchronization ovulation program. nelore heifers (n = 903), primiparous lactating cows (n = 338) and multiparous lactating cows (n = 1,223) were synchronized using a conventional protocol of estradiol / p4-based fixed - time artificial insemination (ftai). thirty days after ultrasonography, females who failed the first ftai were resynchronized with the same hormonal protocol prior to a second ftai. the pregnancy status of each cohort was evaluated by ultrasonography 30 days after each ftai. the average conception rate after the first ftai and resynchronization was 80.5%. heifers had a higher conception rate (85%) than primiparous (76%) or multiparous cows (78% ; p = 0.0001). the conception rate after the first ftai was similar among heifers (57%), primiparous cows (51%) and multiparous cows (56% ; p = 0.193). after the second ftai, heifers exhibited a higher conception rate (66%) than primiparous or multiparous cows (51% ; p = 0.0001). these results demonstrate the feasibility of resynchronization in large beef herds for providing consistent pregnancy rates in a short period of time. we also demonstrated that ovulation resynchronization 30 days after ftai is particularly effective for heifers, providing a conception rate of up to 66%. |
the national eye institute estimates that there are 1.5 million surgeries for cataracts each year in the u.s. a common clinical scenario in a percentage of patients undergoing cataract extraction is receiving treatment for co - morbidities like glaucoma with hypotensive lipids. we report a case of a 59-year - old man who developed recurrent cystoid macular edema (cme) with three separate trials of prostaglandin analogs following uncomplicated phacoemulsification with intraocular lens implantation. currently, we have no prospective, blinded - control trials to better define the ideal perioperative strategies in this patient population. our patient has a history of advanced pigmentary glaucoma in both eyes, which was controlled preoperatively for many years with xalatan (latanoprost), cosopt (timolol / dorzolamide) fixed combination, and alphagan (brimonidine) 0.1% purite ou. the patient developed visually significant cataracts in both eyes with visual acuity dropping below 20/40 ou prior to surgery. he had uncomplicated cataract extraction with iol implant od, followed one month later by os. his best corrected visual acuity in each eye was 20/20 ou by one week post op. his immediate post - op regimen was avelox (moxifloxacin) qid for one week, diclofenac qid for one month, and pred forte (prednisolone) 1% qid for one week and then tapered off over the next three weeks. our patient first developed cme in his right eye 4-months post - cataract extraction, when he presented with complaints of blurry vision and central distortion. after 1-month, his bcva and intraocular pressure od were 20/70 and 16 mm hg, respectively. over the next two months while off the xalatan (latanoprost), the patient 's cme improved, and visual acuity improved to 20/30 + 2, but intraocular pressure climbed to 28 mmhg. within three weeks of initiating lumigan (bimatoprost) in an effort to control his increased iop his cme symptoms recurred for a second time, and his bcva dropped again to 20/60. the patient 's cme was again re - treated with a combination of topical diclofenac qid, and pred forte (prednisolone) 1% qid. his bcva after resolution of the second episode of cme was 20/25 and remained stable for 13 months. for one year after the second episode of cme, the iop remained in the borderline acceptable range on alphagan (brimonidine) 0.1% purite and cosopt (timolol / dorzolamide) fixed combination ou. he became progressively worse when he presented with an iop of 28 mmhg, at which time a trial of travatan (travoprost) was attempted. travatan (travoprost) was the third prostaglandin therapy to date that seems to have triggered cme in our pseudophakic patient and was again discontinued. the patient 's third episode of cme eventually resolved with the prior treatments, and his bcva stabilized at 20/25. his iop at the time of this report is controlled with cosopt (timolol / dorzolamide) fixed combination and alphagan (brimonidine) 0.1% purite. cme is a painless condition in which swelling or thickening occurs of the central retina (macula) and is usually associated with blurred or distorted vision. the primary cause of cme depends on the underlying disease process, but most pathways eventually lead to vascular instability and breakdown of the blood - retinal barrier. the mller cells in the retina become overwhelmed with fluid leading to their lysis. this results in an accumulation of fluid in the outer plexiform and inner nuclear layers of the retina. endogenous prostaglandins (pgs) are known to modulate normal cell function as well as inflammatory response. the conventional route of aqueous humor flow is through the trabecular meshwork, schlemm 's canal and episcleral vessels. it is thought that pgs may regulate uveoscleral outflow by metalloproteinases- (mmp)-mediated alterations in the ciliary muscle in extracellular matrix metabolism [24 ]. it has also been shown that xalatan (latanoprost) induces cystoid macular edema (cme) in patients in the early postoperative period of cataract surgery as well as numerous case reports documenting cme with xalatan (latanoprost) use [57 ]. halpern and pasquale documents thirty - four eyes of thirty - two patients with documented cme in aphakia and pseudophakia. structurally, xalatan (latanoprost), travatan (travoprost), and lumigan (bimatoprost) are all compounds related to prostaglandin 2. both xalatan (latanoprost) and travatan (travoprost) are pharmacologically classified as prostaglandin analogs, but lumigan (bimatoprost) is considered to be a prostamide because it is an amide rather than an ester compound. xalatan (latanoprost) is an isopropyl ester prodrug of 17-phenyl substituted prostaglandin f2 that effectively lowers iop by enhancing uveoscleral outflow without significantly affecting other parameters of aqueous humor dynamics. however, the association between ocular hypotensive lipids and cystoid macula edema continues to be subject of debate. it has been suggested that it is unlikely topical xalatan (latanoprost) induces cystoid macula edema in eyes with a normally functioning blood - ocular barrier. interestingly, our patient, who had been uneventfully treated with xalatan (latanoprost) for several years, developed cystoid macular edema that worsened with rechallenge of lumigan (bimatoprost) and travatan (travoprost), even more than one year after uncomplicated anterior segment surgery. cystoid macular edema associated with xalatan (latanoprost) has been reported after uncomplicated cataract surgery, but it is an uncommon complication. in one retrospective review of a cohort of patients after uneventful phacoemulsification, 3% developed clinically significant cme. it has also been associated with lumigan (bimatoprost) in an eye with high - risk profile. to our knowledge, this case represents the only report in the literature that definitively identifies prostaglandin analogs as the inciting agent in recurrent cme. optical coherence tomography (oct) is helpful in establishing a diagnosis and in measuring the therapeutic response. medical treatment modalities include corticosteroids, which directly inhibit the enzyme phospholipase, blocking the formation of prostaglandins. they are considered the primary treatment of cme in many instances, specifically in the treatment of cme secondary to uveitis. however, corticosteroids have many systemic and ocular adverse effects, and some patients become intolerant to them as a result. nsaids, like diclofenac (voltaren), inhibit the enzyme cyclooxygenase and can be used in the prevention and treatment of cme. nsaids are sually administered topically for approximately 3 - 4 months and on an asneededbasis. in a prospective, double - masked, study of indomethacin versus placebo in the treatment of patients undergoing cataract extraction in addition, treatment of cme with steroid and nsaids has been shown to be better than nsaids alone. despite multiple case reports of individual prostaglandin analogues being suggested as the cause of cme, there is currently no consensus or recommendation regarding the use of these medications in the perioperative period. remissions and exacerbations of macular edema can result in photoreceptor damage with permanent impairment of vision. definitive conclusions about causal relationships can not be made without well - designed, prospective clinical trials addressing this issue given the large number of patients undergoing cataract extraction every year in the u.s. while being treated for glaucoma. more research in this area is important to understand if a causal relationship between the use of pg analogs and the occurrence of cme is more than anecdotal associations in the case reports. caution might be advised when using pg analogs in eyes with risk factors for the development of cme. | to our knowledge, we are reporting the first case of a 59-year - old man who developed recurrent cme with three separate trials of three different prostaglandin class drugs following uncomplicated phacoemulsification with intraocular lens implantation. despite multiple reports of individual prostaglandin (pg) analogues being suggested as the cause of cme, there are no recommendations regarding withholding these medications in the perioperative period. our patient first developed cme od 4-months post uncomplicated cataract extraction. xalatan (latanoprost) had been restarted after surgery and discontinued at onset of cme. while off xalatan (latanoprost), the patient 's cme resolved, but his iop rose. the patient was started on lumigan (bimatoprost) to control the iop, but within weeks his cme recurred. the patient 's cme was again treated and his iop remained acceptable, but then progressively increased. travatan (travoprost) was attempted, but he presented with a third round of cme. definitive conclusions about causal relationships can not be made without well - designed, prospective clinical trials addressing this issue. |
the fibrous osseous lesions of the jaw represent a diverse group of entities, and aree characterized by replacement of normal bony architecture by fibrous connective tissue matrix with varying degrees of osteoid, immature and mature bone. the second edition of the who classification of odontogenic tumor defines juvenile ossifying fibroma (jof) as a lesion consisting of cell - rich fibrous tissue containing bands of cellular osteoid without osteoblastic rimming with trabeculae of more typical woven bone. the lesions having this morphology have been reported as young ossifying fibroma, juvenile active ossifying fibroma, aggressive ossifying fibroma, trabecular desmo - osteoblastoma and active fibrous dysplasia.[24 ] jof occurs predominantly but not exclusively in children. although it can occur anywhere in the skeleton, its highest incidence is in the facial bone.[46 ] it can expand the involved bones, leading to facial asymmetry. depending on the site, symptoms such as pain, paresthesia, malocclusion, sinusitis, proptosis, etc. can also occur due to the swelling.[68 ] root displacement is common and resorption, although rare, can occur.[68 ] the lesion can cause expansion as well as perforation. radiographically, they appear radiolucent, radioopaque or mixed radiolucent radioopaque with a well - defined sclerotic border.[357 ] two histopathologic variants of ossifying fibroma of craniofacial skeleton have been described trabecular juvenile ossifying fibroma (trjof) and psammomatoid juvenile ossifying fibroma (psjof). a 15-year - old female patient reported to our out patient department (opd) with a painless swelling on the right side of the face since 2 1/2 years. the patient related this swelling to extractions done in that region 2 years back at a private clinic. extraoral examination showed facial asymmetry due to a diffuse swelling on the right side of the face [figure 1 ]. the swelling measured approximately 2 cm 3 cm in size and extended from the ala of nose till just in front of the tragus of the right ear. superoinferiorly, it extended from the right infraorbital region to 23 cm short of the inferior border of the mandible. facial asymmetry due to a diffuse swelling on the right side of the face extending from the ala of nose to posterior in front of the tragus of the right ear intraoral examination revealed a solitary, well - defined swelling obliterating the vestibule and extending from the right maxillary canine region (13) to the right second molar region (17) posteriorly [figure 2 ]. on palpation, the swelling was hard and tender, causing expansion of the buccal cortical plates. a solitary, well - defined swelling obliterating the vestibule and extending from the right maxillary canine region (13) to the right second molar region (17) a provisional diagnosis of monostotic fibrous dysplasia of right maxilla was made, with the differential diagnosis of ossifying fibroma. radiological investigations included orthopantomogram, maxillary lateral occlusal projection, paranasal (pns) sinus view and computed tomography (ct) scan. radioopaque lesion in the right maxillary premolar and molar regions giving a ground glass appearance [figure 3 ]. pns view showed an ill - defined radioopacity occupying the floor of the maxillary sinus [figure 4 ]. ct scan showed a hyperdense focal expansile area involving the right maxillary bone, premolars and molars, compressing the lateral wall of the right maxillary sinus with thinning of the cortex [figures 5 ]. radioopaque lesion in the right maxillary premolar and molar region giving a ground glass appearance paranasal view showing an ill - defined radioopacity occupying the floor of the maxillary sinus axial computed tomography scan showing a hyperdense focal expansile area involving the right maxillary bone, premolars and molars compressing the lateral wall of the right maxillary sinus with thinning of the cortex the patient was operated under general anesthesia. because the lesion was slowly growing, a recontouring procedure was performed involving multiple osteotomies. hematoxylin and eosin (hande)-stained sections showed proliferative cellular connective tissue stroma encasing plump and irregular cells [figure 6a ], comprising of mineralized material in the form of trabeculae of woven bone [figure 6b ]. post - operative healing was uneventful [figure 7 ] and the patient is under regular follow - up considering the high recurrence rates of this neoplasm. hematoxylin and eosin - stained section (10) showing proliferative cellular connective tissue stroma encasing plump and irregular cells hematoxylin and eosin - stained section (10) comprising of mineralized material in the form of trabeculae of woven bone hematoxylin and eosin - stained section (40) showing trabeculae without osteoblastic rimming post - operative picture of the patient a 15-year - old male patient reported to our opd with the chief complaint of a gradually increasing asymmetry of the face. clinical examination revealed a moderately large left facial mass over the left molar region [figure 8 ]. intraoral examination revealed gross expansion of the left maxillary alveolar process extending from the 12 region to the 17 region posteriorly, obliterating the buccal vestibule [figure 9 ]. on palpation, the swelling was hard and tender causing expansion of the buccal cortical plates. maxillary cross - sectional occlusal radiograph revealed expansion of the cortical plates [figure 10 ]. hande - stained sections showed a similar histopathological picture as in case 1 [figures 11 and 12 ], and were again suggestive of trabecular variant of jof. the lesion was treated by complete surgical excision. a moderately large left facial mass over the left molar region gross expansion of the left maxillary alveolar process extending from the 12 region to the 17 region posteriorly, obliterating the buccal vestibule maxillary cross - sectional occlusal radiograph showing cortical plates expansion two bits of excised gross specimen sent for histopathological examination hematoxylin and eosin - stained section (10) showing proliferative cellular connective tissue stroma encasing plump cells comprising of mineralized material in the form of trabeculae of woven bone without osteoblastic rimming a 15-year - old female patient reported to our out patient department (opd) with a painless swelling on the right side of the face since 2 1/2 years. the patient related this swelling to extractions done in that region 2 years back at a private clinic. extraoral examination showed facial asymmetry due to a diffuse swelling on the right side of the face [figure 1 ]. the swelling measured approximately 2 cm 3 cm in size and extended from the ala of nose till just in front of the tragus of the right ear. superoinferiorly, it extended from the right infraorbital region to 23 cm short of the inferior border of the mandible. facial asymmetry due to a diffuse swelling on the right side of the face extending from the ala of nose to posterior in front of the tragus of the right ear intraoral examination revealed a solitary, well - defined swelling obliterating the vestibule and extending from the right maxillary canine region (13) to the right second molar region (17) posteriorly [figure 2 ]. on palpation, the swelling was hard and tender, causing expansion of the buccal cortical plates. a solitary, well - defined swelling obliterating the vestibule and extending from the right maxillary canine region (13) to the right second molar region (17) a provisional diagnosis of monostotic fibrous dysplasia of right maxilla was made, with the differential diagnosis of ossifying fibroma. radiological investigations included orthopantomogram, maxillary lateral occlusal projection, paranasal (pns) sinus view and computed tomography (ct) scan. radioopaque lesion in the right maxillary premolar and molar regions giving a ground glass appearance [figure 3 ]. pns view showed an ill - defined radioopacity occupying the floor of the maxillary sinus [figure 4 ]. ct scan showed a hyperdense focal expansile area involving the right maxillary bone, premolars and molars, compressing the lateral wall of the right maxillary sinus with thinning of the cortex [figures 5 ]. radioopaque lesion in the right maxillary premolar and molar region giving a ground glass appearance paranasal view showing an ill - defined radioopacity occupying the floor of the maxillary sinus axial computed tomography scan showing a hyperdense focal expansile area involving the right maxillary bone, premolars and molars compressing the lateral wall of the right maxillary sinus with thinning of the cortex the patient was operated under general anesthesia. because the lesion was slowly growing, a recontouring procedure was performed involving multiple osteotomies. hematoxylin and eosin (hande)-stained sections showed proliferative cellular connective tissue stroma encasing plump and irregular cells [figure 6a ], comprising of mineralized material in the form of trabeculae of woven bone [figure 6b ]. post - operative healing was uneventful [figure 7 ] and the patient is under regular follow - up considering the high recurrence rates of this neoplasm. hematoxylin and eosin - stained section (10) showing proliferative cellular connective tissue stroma encasing plump and irregular cells hematoxylin and eosin - stained section (10) comprising of mineralized material in the form of trabeculae of woven bone hematoxylin and eosin - stained section (40) showing trabeculae without osteoblastic rimming post - operative picture of the patient a 15-year - old male patient reported to our opd with the chief complaint of a gradually increasing asymmetry of the face. he had no significant medical, dental or family history. clinical examination revealed a moderately large left facial mass over the left molar region [figure 8 ]. intraoral examination revealed gross expansion of the left maxillary alveolar process extending from the 12 region to the 17 region posteriorly, obliterating the buccal vestibule [figure 9 ]. on palpation, the swelling was hard and tender causing expansion of the buccal cortical plates. maxillary cross - sectional occlusal radiograph revealed expansion of the cortical plates [figure 10 ]. hande - stained sections showed a similar histopathological picture as in case 1 [figures 11 and 12 ], and were again suggestive of trabecular variant of jof. a moderately large left facial mass over the left molar region gross expansion of the left maxillary alveolar process extending from the 12 region to the 17 region posteriorly, obliterating the buccal vestibule maxillary cross - sectional occlusal radiograph showing cortical plates expansion two bits of excised gross specimen sent for histopathological examination hematoxylin and eosin - stained section (10) showing proliferative cellular connective tissue stroma encasing plump cells comprising of mineralized material in the form of trabeculae of woven bone without osteoblastic rimming jof is an uncommon benign fibroosseous tumor occurring within facial bones in 85% of the patients. these lesions are generally more defined, but may displace the teeth and invade the adjacent bone. minority of the cases, particularly in children (below 15 years), exhibit rapid growth and a tendency to recur and thus have been named jof. gender predilection has been a matter of controversy, with some authors claiming no predilection for either sex, whereas johnson. found a higher incidence in females and el - mofty reported a male predilection. jof has been thought to arise from differentiation of mesenchymal cells of periodontal ligament, multipotent precursor cells, forming into fibrous tissue, cementum or osteoid. lawton. suggested that they perhaps originate from maldevelopment of tissue generating bony septa, between roots of the molar teeth. association of new tumor suppressor gene (hrpt2) mutation has been reported by pimenta., suggesting that the lesion could arise as a result of heploinsufficiency of the particular gene. because of its distinct histological features, it has been recognized as a separate histopathological entity among the fibroosseous group of lesions. el mofty in his study identified two histopathological variants of jof trjof and psjof. this trabecular variant of jof was previously used by reed and hagy in 1965 under jof, who reported two cases one in the maxilla and the other in the mandible. makek in 1983 published the largest series of tumor to date and reviewed 24 cases, 15 from the world literature and nine of his own, from files of the zurich university hospital and called the tumor trabecular desmo - osteoblastoma. he showed a slight male predilection (1.3:1), with maxilla being more commonly affected. reported 10 cases showing a mean age of 11.8 years, again showing a male dominance, with 60% of the cases occurring in the maxilla. dehner, and slootweg and miller reported a few more cases of trjof. both our cases occurred in the maxilla, one in a male patient and the other in a female patient. it might expand the facial bone, causing facial asymmetry as seen in the present cases. after reviewing the literature, it was concluded that the patients age ranged from 2 to 33 years, with an average age range of 8 1/2 to 12 years and with a male dominance. in the maxilla, slow growth has also been reported in a few cases.[469 ] histopathological examination of the trjof shows a well - defined but encapsulated lesion that infiltrates the surrounding bone. progressive calcification of the osteoid results in anastomosing trabeculae of immature woven bone, as seen in both our cases. cystic degeneration and abc formation have been described in a few cases.[2469 ] presence of proliferating cellular connective tissue with plump cells is suggestive of the aggressive nature of this neoplasm, as seen with the present two cases. both the tumor illustrate differences in clinical and dermograpic presentation as shown in table 1 (adapted and modified). differences in clinical and dermograpic presentations of psammomatoid juvenile ossifying fibroma (psjof) and trabecular juvenile ossifying fibroma (trjof) (adapted and modified). radiographically, trjof shows unilocular or multilocular radiolucency with a variable degree of calcification manifesting as fine specks and occasionally producing ground glass appearance. the tumor may reveal cortical thinning and perforation with root resorption and displacement of involved teeth.[57 ] ct findings depend on the stage of development and amount of mineralized matrix present. ct attenuation levels have been reported to range from 34 to 513 hounsfield (hu) depending on the fibrous tissue and bone content. ct findings have been described in only two reports in the radiology literature and in few surgical reports. on ct, the main differential diagnosis would be adult form of ossifying fibroma, fibrous dysplasia and cementoosseous dysplasia. jof will show ct changes similar to the conventional form, but, sometimes, may appear more aggressive with cortical destruction. fibrous dysplasia has a typical ground glass appearance, and expands the bone throughout its length and has poorly defined borders that blend with the surrounding normal bone. cementoosseous dysplasia (apical, focal and florid) is a self - limiting localized lesion present exclusively in the tooth - bearing area of the jaws. ct greatly increases the diagnosis and treatment plan by giving an accurate site, extent of the lesion and size of the tumor mass. on magnetic resonance imaging they reflect an intermediate signal intensity on t1-weighted imaging and an hypointense signal on t2-weighted imaging with moderate enhancement following iv administration of contrast on t1-weighted imaging. fibroosseous lesions present a diagnostic dilemma owing to overlapping clinical and histopathological findings. a differential diagnosis of fibrous dysplasia, malignant bony tumors, aneurysmal bone cyst, central giant cell granuloma, osteogenic sarcoma, osteoblastoma, calcifying odontogenic cyst, adenomatoid odontogenic tumor and primordial cysts (keratocyst) should be considered. fibrous dysplasia can be ruled out as it typically blends with normal bone at the margin of the lesion and has less cellular stroma, and its osteoid does not exhibit osteoblastic rimming and large amount of lamellar bone is found rather than woven bone. central giant cell granuloma occur more commonly in the anterior mandible, resulting in generally painless expansion of bone and appearing radiographically as unilocular or multilocular radiolucent defects with well - delineated, non - corticated margins. the radiographic features of osteosarcoma are orthoradial striations, destruction of cortices with an outgrowth of the soft tissue component, generalized widening of the periodontal ligament spaces and destruction of the lamina dura. vascular tumors like central hemangioma occur mainly in children and young adults and should also be taken in the differential diagnosis of jof. av malformations also exhibit rapid growth, but usually display thrills or bruits on examination. treatment protocol ranges from simple curettage and curettage with peripheral ostectomy to block resection and segmental resection of mandible. radiotherapy has been proven ineffective and contraindicated due to the increased incidence of malignant transformation ranging from 0.4% to 0%. despite the aggressive behavior, because of the aggressive nature of this entity and its high recurrence rate (3050%), jof should be treated like a locally aggressive neoplasm, very much like an ameloblastoma. surgical resection, rather than conservative curettage, is therefore the preferred line of treatment. jof is a rare fibroosseous neoplasm found in the young age group, which is considered locally aggressive than the conventional form and spreads quickly. therefore, it is important to diagnose the lesion early and correlate all available clinical, radiological, ct scan and histologic data for better management. | juvenile ossifying fibroma (jof) is a rare controversial fibroosseous lesion affecting the craniofacial skeleton and occurring commonly in children and young adults. it is highly aggressive and has a high tendency to recur. it is distinguished from the adult variant of ossifying fibroma on the basis of age, site, clinical behavior and microscopic appearance. because of its high recurrence rate, which is 3058%, complete excision is essential. early diagnosis will circumvent the necessity of radical treatment. we report a rare case of trabecular jof of maxilla where a computed tomography scan was taken to further support the characteristic feature of the lesion. |
transurethral resection of the prostate (turp) is generally considered the gold standard for the surgical treatment of benign prostate hyperplasia (bph).1 however, laser technology has also been used to treat lower urinary tract symptoms (luts) secondary to bph for more than 15 years.2,3 increasingly, laser therapy is being considered for the surgical management of bph of any size, as an alternative to turp.3,4 several different initial laser techniques have been introduced to make prostatectomy procedures safer and more effective. such techniques include use of high - powered 120 w (greenlight hps ; american medical systems, minnetonka, mn, usa), thulium (tm), diode, holmium, and vela lasers. techniques consist of coagulation (photoselective vaporization of the prostate [pvp ]), vaporization (pvp and diode), resection, and enucleation (tm), depending on the wavelength, power, and type of laser emission. these methods have proven safe among patients with a high risk of bleeding (anticoagulant users, those with a tendency for bleeding, or those with an american society of anesthesiology [asa ] score > 3).5,6 laser prostatectomy is also superior to turp with regard to reducing blood transfusions and the length of hospital stay ; however, it results in a higher reoperation rate.5,7 advanced laser techniques have been proposed as an alternative to turp ; however, current lasers and laser techniques differ considerably in terms of handling and their interaction with tissue. the study reported here aimed to elucidate the differences between laser systems and help urologists critically evaluate the effectiveness of each system in the treatment of luts due to bph. our results provide a valuable guide to assist urologists in selecting the most suitable laser option for each patient. the study enrolled 741 patients between january 2005 and december 2011 at chang gung memorial hospital - linko. patients with postoperative prostate pathology showing malignancies were excluded (24 patients, 3.2%). surgical treatment was initiated according to bph guidelines provided by the european association of urology.8 laser surgery in each group was performed or supervised by a single surgeon who possessed expertise in laser therapy. recorded parameters included age, history of antiplatelet or anticoagulant drugs, international prostatic symptom score (ipss), digital rectal exam, prostate - specific antigen (psa) level, prostate volume, and transitional - zone volume determined by transrectal ultrasound. in addition, uroflow studies were conducted at 3, 6, 12, and 24 months before and after the operation. postoperative evaluation focused on peak - flow rate (qmax) and post - voiding residual urine, the occurrence of acute urine retention, and whether a foley catheter was inserted prior to the operation. we also recorded asa score, duration of operation, length of catheterization, length of hospital stay, as well as intraoperative, early, and late complications. early operative complications (occurring within 30 days of surgery) included acute urine retention requiring re - catheterization, hematuria requiring surgical intervention, and urosepsis. late complications included bladder neck contracture or a urethral stricture that required secondary surgical intervention. pvp is performed using a high - powered 120 w (greenlight hps) laser system and a side - firing addstat fiber (american medical systems) with a core diameter of 600 mm. power settings were increased from 100 to 120 w after tissue was found to become resistant to vaporization.4 conversely, the tm laser operates at a wavelength of 2,000 nm and is delivered as a continuous wave. tm lasers enable the complete absorption of laser energy in water, and due to their slightly shorter wavelength, the depth of penetration is only 0.25 mm. nonetheless, tm lasers are suitable for transurethral vaporization, resection, or enucleation of the prostate. the operating wavelength of 980 nm is near the infrared electromagnetic spectrum, and is therefore easily absorbed by water and hemoglobin. the mean standard deviation of all continuous measures and scores was recorded at baseline and during all follow - up visits. statistical analysis was performed using analysis of variance or student s t - test to produce continuous data approximating normal distribution. continuous variables were compared between treatment groups using a second student s t - test, with p - values 3, higher psa level, prostate volume, and catheterization prior to the operation (p=0.01). most patients taking warfarin and heparin who had a prostate weighing 60 g underwent treatment with a diode laser. the remaining patients were assigned to tm enucleation laser therapy. additionally, the vast majority of patients who were given antiplatelet therapy (aspirin and/or clopidogrel) underwent diode laser therapy (p=0.001). urinary retention rates were as follows : greenlight hps group, 31.1% ; tm group, 38% ; and diode group, 48.6%. the average length of hospital stay was 2.5 1.4 days for the hps group, 2.5 + 1.0 days for the tm group, and 3.1 + 1.2 days for the diode group (p0.05). all groups (figure 1) showed significant improvements in terms of post - void residual urine volume, qmax, and psa level. the psa levels are dramatically decreased after surgery, also indicated improvements for at least 1 year postoperatively. logistic regression results revealed that most cases of induced comorbidity presented significant differences in prostate volume (hazard ratio [hr ] = 1.03 ; 95% confidence interval [ci ] : 1.011.05 ; p=0.003) and catheterization before the operation (hr = 2.92, 95% ci : 1.167.36 ; p=0.023). other factors, including age, incidence of diabetes mellitus, hypertension, and thrombocytopenia, as well as medical record post - turp, ipss score, psa level, and operative time, were not associated with complications in any of the three laser groups. in the tm group, catheterization before the operation presented a higher overall complication rate than that among patients who did not undergo catheterization (p=0.023). additionally, in the tm group, a 1 g increase in prostate volume did not present a significant increase in complication rates, but a prostate volume exceeding 100 g was associated with a significantly higher surgical morbidity rate (table 4). laser prostatectomy for the treatment of luts provides outcomes and lower complication rates at least equal to those obtained with turp.7 however, the idea that turp should be replaced by laser therapy as the gold standard is still not widely accepted due to the lack of studies on a large number of cases. we found that laser prostatectomy resulted in relatively low rates of intraoperative and perioperative complications. we also observed that patients taking aspirin or warfarin had a higher rate of perioperative bleeding compared with those who did not take these drugs ; however, withholding these medications resulted in increased rates of cardiovascular and cerebrovascular complications.6,8 nonetheless, laser prostatectomy was found to be a useful alternative to turp in treating patients taking antiplatelet and anticoagulation drugs.911 patients undergoing diode laser therapy did not require postoperative blood transfusions. the re - catheterization rate was between 4.3% and 20.0%.12,13 patients in the diode laser group had a re - catheterization rate of approximately 17%. rieken reported that among patients who underwent diode laser treatment, 9.6% of those with bladder neck obstruction required reoperation, compared with 3.6% among those who underwent turp.14 in addition, a urethral stricture developed in 5.5% of those undergoing diode laser treatment versus 0.0% undergoing turp.14 the current study revealed higher complication rates in the diode laser group but no significant differences among the three groups (p=0.237). the blood - loss - requiring - blood - transfusion rate has been reported as 0.0%2.2%.14 in our study, blood transfusion was required in one patient undergoing pvp treatment. in previous reviews of early complications, 1.1% of patients required re - catheterization.15,16 reported reoperation rates have been lower than 2.2%.17,18 in the current study, no statistically significant differences were observed between these three groups. as far as we are aware, this is the first study to outline the differences between laser prostatectomy techniques in terms of handling and operative outcome. this paper has provided necessary information to help urologists differentiate between laser systems and critically evaluate the role of these techniques in the treatment of luts due to bph. this study compared the efficacy of three laser techniques used to treat luts due to bph. pvp, a coagulation - based technique, has durable efficacy for prostates weighing 60 g and among patients with thrombocytopenia or who have a tendency for bleeding. in our study, these techniques were found to be equally safe and effective in the treatment of bph without any severe drawbacks as long as patients were selected according to risk factors. however, future research should use higher quality data to further evaluate and compare types of laser treatment to confirm our results. | purposeendoscopic lasers have become a treatment option for benign prostate hyperplasia (bph). the study reported here sought to elucidate the benefits and drawbacks of different laser systems in the treatment of patients with bph.methodsthe study enrolled 741 patients diagnosed with lower urinary tract symptoms secondary to bph during the period january 2005 to december 2011. the techniques used in the study were photoselective vaporization of the prostate, thulium laser prostatectomy, and diode laser prostatectomy. patients were assigned to one of three groups according to the type of laser treatment they received. outcomes were evaluated using the international prostate symptom score (ipss), quality of life, maximal urinary flow rate, post - voiding residual urine volume, and prostate - specific antigen (psa) level.resultsthe baseline characteristics of patients who received diode laser prostatectomy show a significant elevated risk and high american society of anesthesiology score (p=0.001). operative time and catheter removal time differed significantly between the three groups (p=0.001). no cases were converted to transurethral resection of the prostate intraoperatively due to bleeding (p=0.142). among the three groups, there were no significant differences in maximal flow rate, lower post - void residual urine, and postoperative psa level during the entire follow - up period (p<0.05). further, no significant differences in postoperative ipss, quality of life, or bladder neck contracture (p=0.23) were observed. however, a significant difference was observed with regard to prolonged use of foley catheters and prolonged hospital stay among patients in the diode laser group (p=0.001).conclusionlaser prostatectomies are effective in dealing with lower urinary tract symptoms. early subjective functional results (maximal flow rate, ipss, and post - void residual urine) appeared the same as those obtained following laser prostatectomy. thus, it appears that lasers are safe and effective as long as the patients are carefully selected for treatment. |
it has become more frequent since the introduction of national bowel cancer screening programs, with 36,460 procedures performed in the first 3 years after their establishment 1. to maximize efficacy in both screening and non - screening cases, sedation traditionally has been used to minimize technical difficulties 2 and studies demonstrate improved cecal intubation rates with it 3 and reduced patient anxiety about subsequent colonoscopy 4. sedation is not without risk as fewer than 1 % of patients experience cardiovascular problems and respiratory distress is seen in up to 8 of every 1000 patients undergoing the procedure 5. sedation also places social and functional demands upon patients and, depending on the hospital s protocol, may require dedicated recovery time and an escort on discharge 6. a study of screening colonoscopy at a university center reported a median of 20 minutes for the procedure and 21 hours for preparation, travel, and recovery 7. recently, sedation was found to have no effect on polyp or adenoma detection rate 3 while sedation - less procedures may reduce the cecal intubation rate 8, most likely from the discomfort associated with air insufflation 9. however, pain may not be a limiting factor for intubation rates as petrini. showed that of the patients offered sedation on demand, 81 % underwent complete procedures without a sedative 10. the aim of this study is to compare the level of discomfort in patients who underwent non - sedated colonoscopies (nsc) to those with sedation (sc), and to determine the effect on polyp detection and cecal intubation rates. we also assessed the effects of anaesthetic risk, indications, endoscopists, and pathology on outcomes. the prospectively accrued database (unisoft) of all colonoscopies performed at three district general hospitals in the uk between july 2011 and july 2012 was retrospectively analyzed. sedation was routinely offered to all patients, however, 194 declined all medication. throughout the procedure, patient comfort scores were obtained by two independent nurses using modified glasgow comfort score descriptors, as outlined by the nhs bowel cancer screening programme (england, uk) 11 (table 1). staff at the three units are certified by the joint advisory group on gi endoscopy (jag, uk). they performed colonoscopies in accordance with jag s guidelines for bowel preparation, peri - procedure care, and recovery. anaesthetic risk was assessed using the american society of anesthesiologists physical status classification system (asa grade, table 2). details of age, sex, asa grade, comfort scores, complications, cecal intubation rate and polyp detection rates were obtained. an adjusted cecal intubation rate was calculated by including patient discomfort and excluding poor bowel preparation and instrument inadequacy. statistical analysis was performed using chi - squared test, student s t - test or mann - whitney, univariate analysis for odds ratios and a p value less than 0.05 was considered significant. a total of 1694 colonoscopies were performed over a year, of which 194 were without sedation (11 %). table 3 summarizes the key findings for both sc and nsc patients. in both groups there was a male predominance in the nsc group (male to female ratio 1.6:1), which was not demonstrated in the sc group (1:1.2 p < 0.001). there was no gender difference in discomfort levels (p = 0.102). a single endoscopy unit carried out more colonoscopies without sedation than others within the organization (hospital a, p < 0.0001). however, after excluding those who performed less than 100 cases (none of whom carried out nscs), there were no differences in the numbers or rates of incomplete colonoscopies performed by the 10 high - volume endoscopists (p = 0.105). a greater proportion of patients were referred for bleeding (p = 0.034) or frequency of stool (p = 0.036), while fewer were likely to have anaemia (p = 0.014). all asa grades were represented in both groups but there were more grade 3 patients in the nsc group (p = 0.0003). a greater proportion experienced lower levels of discomfort (no discomfort 43 %, minimal 29 %, 13 % mild discomfort, p < 0.001). there were a comparable number of complications ; three cases of mechanical damage to scope and two cases of haemorrhage occurred in the sedated group. significant values were p < 0.05, derived by chi - square test, t - test or mann - whitney. cibh : change in bowel habit only indications with significant p values are shown no significant differences were found for diverticular disease, strictures or radiation proctitis. polyps were found in 51 out of 194 nsc patients (26 %) and comparable to the sc group (23 % p = 0.249). the rates of failed cecal intubation were also similar at 11 % and 10 % respectively (p = 0.624). incomplete colonoscopies in the nsc group were due to inadequate bowel preparation, angulation or fixity of the distal sigmoid, instrument inadequacy, and, in three cases, patient discomfort ; the adjusted cecal intubation rate was 95.4 % (table 4). when compared to sc, nsc offers patients a safe investigation with shorter recovery time without the assistance of an escort and with return to normal daily activities afterwards. anecdotally, nsc facilitates better communication between the endoscopist and the patient, which may assist in positional changes towards successful completion. in our study, all patients were offered sedation at the start, and if they refused, it was offered again if these patients experienced discomfort during the procedure. from our data, we could not determine why patients did not want sedation or why some patients who started without sedation required that treatment when difficulty was encountered. of the 194 nsc patients, there was a male predominance. while colorectal adenoma is more prevalent in men 12, polyp detection rates were similar in both sc and nsc patients. arguably this may suggest under - detection of polyps, but that is less likely because the procedures were performed by jag - accredited and experienced screening endoscopists. in addition, we included patients referred via the suspected cancer or screening pathways, who may have a higher risk for colorectal neoplasia, but a risk adjusted analysis of these factors requires a larger study. as the level of discomfort experienced by patients was recorded at three sites within a single organization, interobserver variability could have affected perception of patient discomfort. however, all units are certified by jag and follow standard protocol for all procedures for recording discomfort. while endoscopist experience is known to affect polyp detection rates 13 14, it was not controlled for in our study. because all endoscopists were jag accredited, it ensured that their skill levels were equal, and therefore, unlikely to influence outcomes. multivariate analysis of our results revealed that patients undergoing nsc were more likely to have more comorbidities as evidenced by higher asa grades. nsc would certainly be a safer option given the risk that sedation poses to cardiovascular stability. we could not determine whether nsc was a patient s or endoscopist s choice, but exploring their motivations might help identify factors that could improve its uptake. we also demonstrated that some hospital units were more likely to use nsc than others, but the decision was not influenced by the endoscopist performing the procedure. hospital factors that may affect decisions for nsc, such as outcome targets, patient information, informed consent, staff preferences and biases, warrant investigation even though they are likely to vary from one institution to another. previous studies have highlighted how discomfort may limit the completion of the procedure, and advocated the benefits of water insufflation 15, sedation on demand (patient controlled), and sedation as needed (endoscopist controlled) 16. overall, we showed that nsc can be completed comfortably with minimal variation in technique, supporting previous findings of success more so in men, and that it had no effect on polyp detection rate 3. successful nsc is less likely with macroscopic inflammation as it was associated with significant discomfort. our cecal intubation rate matched that of previous studies 8 and was similar to that achieved when sedation was used. the adjusted cecal intubation rate was 95.4 %, which satisfies the standard set by the nhs bowel cancer screening programme 11. that suggests that nsc with optimal bowel preparation and adequate instrumentation may achieve the high technical standards required by the screening program. it may also appeal to patients without an escort who are candidates for same - day discharge. a recent study showed that 56.2 % of 964 patients were willing to undergo nsc, and that fear of procedure - related pain was inversely related to procedure acceptance 17. as moderate discomfort was described in only 5 % of cases, our study promotes nsc, or at least sedation on demand, although it critically lacked a post - procedure survey of patients experience. overall, our results confirm that nsc is a practical option for motivated patients who are adequately counselled prior to the procedure. it has the potential to avoid sedation - related complications, particularly in patients with multiple comorbidities, to prevent delay in discharge from hospital, and to allow near immediate return to normal activity. | background and study aims : conscious sedation during colonoscopy minimizes discomfort, improves polyp detection rates, and reduces technical failure, but carries medication - related risks and requires dedicated and costly recovery services. sedation - free procedures may offer a safer alternative. we aimed to compare this group with those receiving sedation to determine differences in patient characteristics, cecal intubation rates, polyp detection rates, discomfort levels and safety in patients for whom anesthesia is high risk. patients and methods : prospectively collected data from all colonoscopies performed over a 1-year period at three district general hospitals were analyzed. conscious sedation was offered to all patients and outcomes in those who refused were compared with outcomes in those who received sedation. results : one hundred ninety - four of 1694 (11 %) colonoscopies were performed without sedation (61 % male, p < 0.001) but rates varied between hospitals. of these, 55 % were american society of anesthesiologists (asa) grade 3 or more and 5 % experienced moderate discomfort, compared to 40 % (p < 0.0001) and 10 % (p = 0.023) respectively of those receiving sedation. they were more likely to have indications of rectal bleeding or frequency of stool and less likely to have anaemia or macroscopic inflammation at colonoscopy. complications, completion. and polyp detection rates were similar in both groups. conclusions : colonoscopy without sedation can be completed successfully in select patients without compromising comfort or polyp detection rates and is safe in those for whom anesthesia is high risk. it is therefore a safe alternative for clinicians concerned about sedation, but the findings suggest that hospital, rather than patient factors, may prevent its uptake. |
multi - frequency bia is described as a tool able to assess total, extracellular and intracellular fluid in humans [14 ], but its ability to diagnose water - loss dehydration has not been previously assessed. water - loss dehydration, signified by raised serum osmolality and due to insufficient fluid intake and sometimes increased output is common after stroke and associated with increased mortality and morbidity [58 ]. dysphagia, depression and concern over continence all increase the risk of dehydration, and stroke is more common in those who are already dehydrated. hydration status can alter rapidly in people not drinking adequately, so monitoring hydration status in this particularly vulnerable group is important especially in non - hospital settings such as residential and nursing homes. serum osmolality, the best reference standard for water - loss (or hypertonic) dehydration is the osmolar concentration or osmotic pressure of serum, reflecting the concentration of dissolved particles per kilogram of serum. serum osmolality reflects the osmolality of intracellular fluid, and thus cell volume, as most cell walls are permeable to water allowing osmolality to equalise between the intra- and extra - cellular fluid. as osmolality is carefully controlled by the body, any change suggests important alterations in cellular hydration. serum osmolality has advantages as a reference standard for dehydration as it can be used alone, and without prior measurement. weight change has been used as a marker for dehydration but requires at least 2 measurements (so can not be used on an unknown patient), and can be masked by changes in other body components (fat and muscle) as well as mimicked or masked by constipation and oedema. thus serum osmolality has the best easily measurable characteristics of a reference standard for water - loss dehydration and is the diagnostic standard against which the accuracy of other measures should be judged. the dehydration council s cut - off points for serum osmolality are specific to water - loss dehydration in older people. serum osmolality of 295300 mosm / kg equates to impending dehydration, and > 300 mosm / kg to current dehydration, definitions used in this study. in clinical practice serum osmolality is often not directly measured, but calculated from the combined concentrations of serum sodium, potassium, glucose and urea, referred to as serum osmolarity ([2na]+[2k]+urea+glucose, all in mmol there is a small difference between measured serum osmolality and calculated osmolarity, known as the osmolar gap (as some components of osmolality are not included in the formula to calculate osmolarity). methods to assess hydration status which do not require blood samples would be helpful for use in those who have had a stroke once they leave hospital, especially in situations where there is no quick and easy access to laboratory facilities such as rehabilitation services, care homes and the community. there is some evidence that signs used to indicate water - loss dehydration in older people such as urinary and physical assessments are unreliable and inaccurate. while single frequency bioelectrical impedance assumes full hydration and so is unable to assess hydration status, multi frequency bioelectrical impedance analysis (mf - bia) unlike single frequency bia, can provide estimates of total body water (tbw), intracellular water (icw), and extracellular water (ecw) volumes and as percentages of body weight. this is because the 5 khz frequency can estimate ecw, while frequencies over 50khz predict tbw (and icw = tbw - ecw). the maltron website states the bioscan 920 - 2s multi - frequency analyser with its unique features is a rapid, non - invasive, inexpensive method for evaluating hydration and nutrition status the advance (sic) circuitry and processing power of the bioscan 920 - 2s allows it to measure extracellular (ecf) and intracellular fluid (icf) volume (see www.maltronint.com/products/bioscan920-2s.php, accessed 6/11/2012). despite this claim of evaluating hydration status, the diagnostic accuracy of mf - bia in diagnosis of dehydration has not previously been published to our knowledge. this study aimed to assess the diagnostic accuracy of mf - bia, to help understand whether mf - bia can be used to monitor hydration status in place of serum osmolality after stroke. this prospective study was carried out in the norfolk and norwich university hospital in the east of england. twenty seven stroke patients admitted within 48 hours of symptom onset were recruited from 1 april to 15 oct 2011. patients were included if older than 17 years, with newly diagnosed stroke (first or recurrent). exclusions included those with severe stroke (national institute of health stroke scale, nihss, score > 30), co - existing terminal illness, expected survival 300 mosm / kg ; serum osmolarity > 300 a paired sample student s t - test was carried out to compare the difference in serum osmolality and calculated osmolarity values overall and stratified by hydration status ; hydrated, impending, and current dehydration. an average was calculated for each two consecutive measurements taken by mf - bia of same variable for use in subsequent calculations. for the one participant where the two consecutive estimates of tbw% varied by > 3% the first data set was used. the internal consistency of mf - bia was assessed by carrying out a reliability analysis of the 2 separate measurements of impedance at 5 khz for each individual. this was repeated for impedance measures at 50 and 100 khz, and the mf - bia equipment calculation of tbw (l). impedance outputs (mean from the two readings) were used to calculate tbw (l) and ecw (l) using equations developed for use in older people (mean age 67, similar to our participants) by vach and visser (as quoted in ritz (22)), and tbw%, ecw% and icw% were calculated as percentages of body weight1. total body water (bw) was calculated using vach equations (19) and used in estimating tbw : where r100 is impedance at 100hz, g is gender (0 for women and 1 for men) and height and weight are measured in meters and kilograms. extracellular water was estimated using equations by visser (as quoted in ritz): where r5 is impedance at 5hz. tbw%, ecw%, icw% and ecw : icw ratio from the internal calculations of the mf - bia equipment, and those calculated from equations derived specifically for older people were each plotted in 22 tables against impending and current serum osmolality and calculated serum osmolarity. these tables were used to calculate sensitivity, specificity, positive predictive value (ppv), negative predictive value (npv), pre- and post - test probability of each for impending and current dehydration. where any of these values were not calculable due to the presence of zeros in the 22 table, 0.1 was added to each cell of the table. as published cut - off points of tbw, ecw and icw for dehydration are not readily available, three arbitrary cut - off points were selected for each measure (tbw%, ecw%, icw% and the ratio). receiver operating characteristic (roc) curves were created for both impending and current dehydration, then additional promising cut - off points (where cut - offs may possibly have both sensitivity and specificity > 60%) were added to fill in the roc curves. at the ends of the roc curve, once either sensitivity or specificity was below 50%, no further outlying points were added. an acceptable cut - off point was considered to be one with both sensitivity and specificity greater than 60% and represented by the point closest to the top left corner of the roc plot. there is no definition of good enough sensitivity and specificity but we chose a minimum of 60% for both as suggesting that the measure was at least promising. for all cut - off points we also calculated positive predictive value (ppv), negative predictive value (npv), and positive and negative post - test probabilities. no sample size calculation was performed as there were no data available previously reporting diagnostic accuracy of mf - bia against serum osmolality. twenty seven participants were a realistic sample given the time frame we were able to use for this study. all statistical analyses were carried out using pasw 18 for windows (polar engineering and consulting, formerly known as spss). mean, standard deviation (sd) and range were presented for continuous and numbers (percentages) were presented for categorical data. percentages of patients diagnosed with impending (serum osmolality 295300 mosm / kg ; serum osmolarity 295300 mosm / l) and current dehydration (serum osmolality > 300 mosm / kg ; serum osmolarity > 300 was carried out to compare the difference in serum osmolality and calculated osmolarity values overall and stratified by hydration status ; hydrated, impending, and current dehydration. an average was calculated for each two consecutive measurements taken by mf - bia of same variable for use in subsequent calculations. for the one participant where the two consecutive estimates of tbw% varied by > 3% the first data set was used. the internal consistency of mf - bia was assessed by carrying out a reliability analysis of the 2 separate measurements of impedance at 5 khz for each individual. this was repeated for impedance measures at 50 and 100 khz, and the mf - bia equipment calculation of tbw (l). impedance outputs (mean from the two readings) were used to calculate tbw (l) and ecw (l) using equations developed for use in older people (mean age 67, similar to our participants) by vach and visser (as quoted in ritz (22)), and tbw%, ecw% and icw% were calculated as percentages of body weight1. total body water (bw) was calculated using vach equations (19) and used in estimating tbw : where r100 is impedance at 100hz, g is gender (0 for women and 1 for men) and height and weight are measured in meters and kilograms. extracellular water was estimated using equations by visser (as quoted in ritz): where r5 is impedance at 5hz. tbw%, ecw%, icw% and ecw : icw ratio from the internal calculations of the mf - bia equipment, and those calculated from equations derived specifically for older people were each plotted in 22 tables against impending and current serum osmolality and calculated serum osmolarity. these tables were used to calculate sensitivity, specificity, positive predictive value (ppv), negative predictive value (npv), pre- and post - test probability of each for impending and current dehydration. where any of these values were not calculable due to the presence of zeros in the 22 table, 0.1 was added to each cell of the table. as published cut - off points of tbw, ecw and icw for dehydration are not readily available, three arbitrary cut - off points were selected for each measure (tbw%, ecw%, icw% and the ratio). receiver operating characteristic (roc) curves were created for both impending and current dehydration, then additional promising cut - off points (where cut - offs may possibly have both sensitivity and specificity > 60%) were added to fill in the roc curves. at the ends of the roc curve, once either sensitivity or specificity was below 50%, no further outlying points were added. an acceptable cut - off point was considered to be one with both sensitivity and specificity greater than 60% and represented by the point closest to the top left corner of the roc plot. there is no definition of good enough sensitivity and specificity but we chose a minimum of 60% for both as suggesting that the measure was at least promising. for all cut - off points we also calculated positive predictive value (ppv), negative predictive value (npv), and positive and negative post - test probabilities. no sample size calculation was performed as there were no data available previously reporting diagnostic accuracy of mf - bia against serum osmolality. twenty seven participants were a realistic sample given the time frame we were able to use for this study. of the 27 included participants 12 (44%) were well hydrated (serum osmolality 275 to 300 mosm / kg). stratified by calculated serum osmolarity 8 (30%) were well hydrated (275 to 300mosm / l) (table 1). 11% (n=3) were receiving a nil - by - mouth feeding regimen because of dysphagia (as they were being fed nasogastrically). one patient was on pureed diet and 19% (n=5) on soft - mashed diets due to slight swallowing difficulties. sixty seven percent (n=18) were on normal oral diets without manipulation of food texture. serum osmolality, osmolarity, age, height and weight were normally distributed (both kolmogorov - smirnov and wilk - shapiro p - values were > 0.05 suggesting that the data came from a normal distribution). cronbach s alpha () was 0.960 for the reproducibility of the two impedance measures at 5 khz (n=27), suggesting excellent internal consistency. cronbach s alpha also suggested excellent internal consistency for impedance at 50 khz, and 100 khz, and tbw (l) (0.974, 0.978 and 0.995 respectively). current dehydration (> 300 mosm / l) diagnosed on serum osmolarity criteria was twice as common as when based on serum osmolality (> 300 mosm / kg), the reference standard (table 2). as calculated osmolarity (in mosm / l) is often used in clinical practice in place of measured osmolality (in mosm / kg) we directly compared the two for individuals. mosm / l while mean measured serum osmolality was 295.5 (7.5) mosm / kg. direct comparison showed that there was a significant difference of 2.72 (95% ci 0.6 to 4.8 ; p=0.014). when stratified by hydration status serum osmolarity was greater than osmolality for hydrated participants (mean difference 4.7, 95% ci 1.1 to 8.2, p=0.02) and those with impending dehydration (mean difference 2.9, 95% ci 0.2 to 5.6, p=0.04) but not for those with current dehydration (mean difference 1.4, 95% ci 7.4 to 4.5, p=0.57). mean serum sodium, potassium, creatinine, urea and glucose values were always higher in those with current dehydration than those who were well hydrated (table 2), but the mean values for impending dehydration were not always between those of hydrated and currently dehydrated groups. there were few clear patterns in tbw%, ecw%, icw% or ecw : icw ratio by serum osmolality or calculated serum osmolarity (table 2). no cut - off point for tbw%, icw%, ecw% or ecw : icw ratio (calculated by the mf - bia equipment) had both a sensitivity and specificity above 60% for impending (supplementary table 1) or current (supplementary table 2) dehydration as assessed against (measured) serum osmolality. figure 1 shows the roc plot for icw% for impending dehydration by serum osmolality and figure 2 shows the roc plot for ecw% for current dehydration by serum osmolality). diagnostic accuracy for tbw%, icw%, ecw% and ecw : icw calculated using the equations specifically developed for older people (rather than those programmed into the mf - bia equipment) compared to serum osmolality resulted in no cut - off points with both sensitivity and specificity > 60% for impending dehydration (supplementary table 3), and one cut - off point for current dehydration (supplementary table 4). tbw% with a cut - off at 46% of body weight, was diagnostic of current dehydration by osmolality with sensitivity of 67% (95% ci 49% to 85%), specificity 62% (95% ci 44% to 80%) (supplementary table 4, figure 3). the positive likelihood ratio (lr) for this cut - off was 1.75 and negative likelihood ratio (lr) was 0.54. no cut - off points for tbw%, icw%, ecw% or ecw : icw as calculated by the mf - bia equipment against calculated serum osmolarity had a sensitivity and specificity above 60% for impending (295 mosm / l serum osmolarity, supplementary table 5) or current dehydration (295 mosm / l, supplementary table 6, suplementary figures 15). diagnostic accuracy for water fractions calculated using the equations for older people against calculated serum osmolarity resulted in one cut - off point with both sensitivity and specificity of at least 60%. tbw% at 47% of body weight was diagnostic of impending dehydration by calculated osmolarity with sensitivity and specificity of 63% (95% ci 45% to 81%) (supplementary table 7 ; figure 4). due to the very limited diagnostic accuracy of tbw%, icw%, ecw% or ecw : icw secondary analyses were considered. as the fluid content of fatty tissue is minimal we hypothesised that diagnostic accuracy might be improved if we separated men and women (as men and women have different proportions of fatty tissue), or analysed total body water as a percentage of fat free mass (lean body weight). analyses were run using total body water data as tbw% was the measure with most diagnostic accuracy in previous analyses. separating out men and women (using tbw as a percentage of total body weight) improved diagnostic accuracy for men for current dehydration (using the ritz equations), and provided a cut - off for impending dehydration (table 3, supplementary tables 9 and 10). the cut - off of 46% for tbw% showed sensitivity of 67% and specificity of 85% for current dehydration while 47% diagnosed impending dehydration (sensitivity 63%, specificity 75%) (supplementary figures 6 and 7). analysing tbw as a percentage of lean body weight provided a potentially useful cut - off for women for impending dehydration at 84% (sensitivity 71%, specificity 75%), but none for current dehydration or for men alone, or men and women combined (supplementary tables 11 and 12, supplementary figures 810). of the 27 included participants 12 (44%) were well hydrated (serum osmolality 275 to 300 mosm / kg). stratified by calculated serum osmolarity 8 (30%) were well hydrated (275 to 300mosm / l) (table 1). 11% (n=3) were receiving a nil - by - mouth feeding regimen because of dysphagia (as they were being fed nasogastrically). one patient was on pureed diet and 19% (n=5) on soft - mashed diets due to slight swallowing difficulties. sixty seven percent (n=18) were on normal oral diets without manipulation of food texture. serum osmolality, osmolarity, age, height and weight were normally distributed (both kolmogorov - smirnov and wilk - shapiro p - values were > 0.05 suggesting that the data came from a normal distribution). cronbach s alpha () was 0.960 for the reproducibility of the two impedance measures at 5 khz (n=27), suggesting excellent internal consistency. cronbach s alpha also suggested excellent internal consistency for impedance at 50 khz, and 100 khz, and tbw (l) (0.974, 0.978 and 0.995 respectively). current dehydration (> 300 mosm / l) diagnosed on serum osmolarity criteria was twice as common as when based on serum osmolality (> 300 mosm / kg), the reference standard (table 2). as calculated osmolarity (in mosm / l) is often used in clinical practice in place of measured osmolality (in mosm / kg) we directly compared the two for individuals. mosm / l while mean measured serum osmolality was 295.5 (7.5) mosm / kg. direct comparison showed that there was a significant difference of 2.72 (95% ci 0.6 to 4.8 ; p=0.014). when stratified by hydration status serum osmolarity was greater than osmolality for hydrated participants (mean difference 4.7, 95% ci 1.1 to 8.2, p=0.02) and those with impending dehydration (mean difference 2.9, 95% ci 0.2 to 5.6, p=0.04) but not for those with current dehydration (mean difference 1.4, 95% ci 7.4 to 4.5, p=0.57). mean serum sodium, potassium, creatinine, urea and glucose values were always higher in those with current dehydration than those who were well hydrated (table 2), but the mean values for impending dehydration were not always between those of hydrated and currently dehydrated groups. there were few clear patterns in tbw%, ecw%, icw% or ecw : icw ratio by serum osmolality or calculated serum osmolarity (table 2). no cut - off point for tbw%, icw%, ecw% or ecw : icw ratio (calculated by the mf - bia equipment) had both a sensitivity and specificity above 60% for impending (supplementary table 1) or current (supplementary table 2) dehydration as assessed against (measured) serum osmolality. figure 1 shows the roc plot for icw% for impending dehydration by serum osmolality and figure 2 shows the roc plot for ecw% for current dehydration by serum osmolality). diagnostic accuracy for tbw%, icw%, ecw% and ecw : icw calculated using the equations specifically developed for older people (rather than those programmed into the mf - bia equipment) compared to serum osmolality resulted in no cut - off points with both sensitivity and specificity > 60% for impending dehydration (supplementary table 3), and one cut - off point for current dehydration (supplementary table 4). tbw% with a cut - off at 46% of body weight, was diagnostic of current dehydration by osmolality with sensitivity of 67% (95% ci 49% to 85%), specificity 62% (95% ci 44% to 80%) (supplementary table 4, figure 3). the positive likelihood ratio (lr) for this cut - off was 1.75 and no cut - off points for tbw%, icw%, ecw% or ecw : icw as calculated by the mf - bia equipment against calculated serum osmolarity had a sensitivity and specificity above 60% for impending (295 mosm / l serum osmolarity, supplementary table 5) or current dehydration (295 mosm / l, supplementary table 6, suplementary figures 15). diagnostic accuracy for water fractions calculated using the equations for older people against calculated serum osmolarity resulted in one cut - off point with both sensitivity and specificity of at least 60%. tbw% at 47% of body weight was diagnostic of impending dehydration by calculated osmolarity with sensitivity and specificity of 63% (95% ci 45% to 81%) (supplementary table 7 ; figure 4). due to the very limited diagnostic accuracy of tbw%, icw%, ecw% or ecw : icw secondary analyses were considered. as the fluid content of fatty tissue is minimal we hypothesised that diagnostic accuracy might be improved if we separated men and women (as men and women have different proportions of fatty tissue), or analysed total body water as a percentage of fat free mass (lean body weight). analyses were run using total body water data as tbw% was the measure with most diagnostic accuracy in previous analyses. separating out men and women (using tbw as a percentage of total body weight) improved diagnostic accuracy for men for current dehydration (using the ritz equations), and provided a cut - off for impending dehydration (table 3, supplementary tables 9 and 10). the cut - off of 46% for tbw% showed sensitivity of 67% and specificity of 85% for current dehydration while 47% diagnosed impending dehydration (sensitivity 63%, specificity 75%) (supplementary figures 6 and 7). analysing tbw as a percentage of lean body weight provided a potentially useful cut - off for women for impending dehydration at 84% (sensitivity 71%, specificity 75%), but none for current dehydration or for men alone, or men and women combined (supplementary tables 11 and 12, supplementary figures 810). although we tried different ways of calculating tbw, icw and ecw, assessed tbw as a percentage of total body weight or lean body weight, separated participants by gender and defined dehydration using both serum osmolality and serum osmolarity only 8 cut - off points had both sensitivity and specificity of at least 60%. the only cut - off with limited diagnostic accuracy for both men and women was observed for tbw% at 46% when calculated using equations developed for older people (sensitivity 67%, specificity 62%) for current dehydration by measured osmolality (> 300 mosmol / kg), but positive and negative likelihood ratios were poor (1.75 and 0.54 respectively). similarly tbw at 47%, only with equations developed for older people, also showed limited diagnostic accuracy (sensitivity 63% and specificity 63%, lr 1.7 and lr 0.6) for impending dehydration as assessed by calculated serum osmolarity (295 mosmol / l). when internal equipment equations for estimating tbw were used no cut - offs were even minimally diagnostic. in secondary analyses cut - offs of tbw as a percentage of total body weight were partially useful in diagnosing impending and water - loss dehydration (by serum osmolality) in men, but none were useful in women. one cut - off for tbw as a percentage of lean body mass was useful in women in diagnosing impending dehydration, but none for current dehydration or in men. in this small population of 27 people with recent strokes, mf - bia did not fulfil its promise as a diagnostic tool for water - loss dehydration. calculated serum osmolarity was not good at predicting those with current dehydration by the reference standard, measured serum osmolality, and using calculated osmolarity resulted in 44% of our population being labelled as having current dehydration, compared to 22% by serum osmolality. research is needed to assess the health impacts of dehydration as measured by changes in serum osmolality, serum osmolarity and weight change, so that we can be sure which is the most useful measure of dehydration to use in future. the limited diagnostic accuracy for current dehydration by osmolality at tbw% of 46% (sensitivity 67%, specificity 62%) using the impedance output from mf - bia to calculate tbw% suggests that only 67 of every 100 people with current dehydration by serum osmolality will be positive using tbw% as the test, meaning that 33 of every 100 with current dehydration will be missed. similarly the specificity of 62% suggests that for every 100 people without current dehydration 62 will have a negative test but 38 will have a positive test2. this is a very high level of false positives and negatives, suggesting that mf - bia is not useful in diagnosing water - loss dehydration independently of other clinical or biochemical data. the test s positive (ppv) and negative (npv) predictive values3 as well as pre and post - test probabilities provide more information on the utility of tbw% at the 46% cut off point. the ppv of 33% (equivalent to the positive post - test probability of 33%) suggests that only 33% of those who are diagnosed as having current dehydration by mf - bia truly have current dehydration by serum osmolality. the npv of 87% is clearly better, meaning that 87% of those diagnosed as not having current dehydration are truly without current dehydration (and this is another way of stating the negative post - test probability of 13%). the positive likelihood ratio (lr) was 1.75 and negative likelihood ratio (lr) 0.544 suggesting that for a person positive for dehydration by this test the odds are 1.75 that dehydration is present compared to 1.00 for a person studies evaluating the utility of mf - bia in diagnosing dehydration in clinical settings are scarce. meta - analysis of studies in which tbw was estimated using bia and validated against the reference standard method, deuterium isotope dilution, found that mf - bia estimated tbw more closely to the reference standard (wmd 0.18l, 95% ci 1.62 to 1.98) than either single frequency (sf) bia (wmd 3.00l, 95% ci 1.43 to 4.57) or bioelectric impedance spectroscopy (wmd 2.80l, 95% ci 1.03 to 4.58). however, there are other indications in the literature that assessment of tbw may not be sufficiently accurate in conditions of change in hydration status and when body compartments are undergoing acute changes. this may be because changes in the ratio of intra- to extra - cellular water, and of acute changes in these compartments, also influence resistivity [2629 ]. this may mean that there are fundamental problems with mf - bia in assessing tbw and so in using mf - bia in predicting hydration status. our results suggesting that mf - bia is not a useful diagnostic tool are in broad agreement with those of olde rikkert 1997. they found that in dehydrated geriatric patients (n=53) the sensitivity of diagnosing dehydration using 100 khz mf - bia measurements was only 14% very poor sensitivity, and sensitivity was not improved when other frequencies were tested. the sensitivity and specificity of mf - bia were similar to that of the much simpler measure, tongue dryness, found in a recent australian study. tongue dryness is easier and quicker to assess, and does not need additional investment or electrical supplies, so would be a more appropriate measure to use in most situations than mf - bia if its diagnostic accuracy is similar. leaving the mathematics of diagnostic accuracy of mf - bia aside and observing data generated by mf - bia also suggested that mf - bia generated outcomes are not coherent with the diagnosis of dehydration. table 2 suggested no significant difference in mf - bia measures between hydrated, impending, and currently dehydrated groups. the intracellular water content reflects information on the state of hydration at the cellular level. cellular hydration status can change within minutes under the effects of stress, nutrients, hormones, and other factors. mf - bia does not appear to usefully reflect changes observed in serum osmolality or osmolarity or to sensitively identify the dehydrated state at the cellular level. haussinger suggested that while a well hydrated cell increases anabolic processes, a dehydrated cell shifts metabolism to catabolism, especially in muscle tissue. if recovery is to occur in a highly stressed patient after stroke, we want to ensure they are in an anabolic rather than a catabolic state. bhalla found that the 30% of their 167 stroke patients who had raised serum osmolality (> 296 mosm / kg) had increased odds of mortality at 3 months (or 2.4, 95%ci 1.0 t 5.9). kelly found that in their 102 acute ischaemic stroke patients raised serum osmolality (> 297 mosm / kg, in 24% of their patients) on day 9 following admission was associated with increased odds of venous thromboembolism (or 4.7, 95% ci 1.4 to 16.3). the maltron website states that the bioscan 920 - 2 multi - frequency analyser with its unique features is a rapid, non - invasive, inexpensive method for evaluating hydration and nutrition status it suggests applications in fluid retention and effects of hydration and dehydration. despite the maltron website reporting that it is quick, safe and easy and no assistance or technical knowledge is required (see www.maltronint.com/popup_pages/bioscan9202.htm, accessed 23/2/2012) we found the machine is not user friendly. without a keyboard, data entry and saving of data are slow and may result in errors and data loss. re - running a second measurement for the same participant requires re - entering all the same information again or the new test overwrites existing data. analysed data are not easily accessible to visual check without downloading the full data set, and there is no warning when unrealistic readings are registered. on - site readout of each variable for each participant was time consuming and unrealistic in an acute stroke unit. in assessing mf - bia the battery was charged between measurements, cables were of appropriate length, and the equipment calibrated for all participants reported in this paper. weight and height were measured by the investigator or by a health professional (not self - reported), mf - bia measurements were carried out at ambient temperature and manufacturer guidelines on positioning of electrodes were followed. before measurements our participants were in a supine position for least 10 min and during measurement they had no contact with any metal object (such as a bed frame). electrodes were attached to the non affected side to record measurements and to skin with no abrasions or deformation that may affect current conductance. none of our participants suffered from edema due to excessive hydration by iv / electrolyte infusion or very low albumin levels. one of the main limitations in a ward setting is the inability of researchers to completely control participants fasting or bladder voidance, but the investigator did ensure that participants had fasted for at least 2 hours before mf - bia measurements were taken and all participants were asked if they would like to void their bladder before measurements commenced. study strengths included the use of serum osmolality and calculated osmolarity as reference standards, a population with high levels of dehydration, and recording serum osmolality and other serum measures (sodium, potassium, glucose, urea) within 20 minutes of mf - bia measurements (enabling us to capture cellular hydration status as evaluated by mf - bia and its coherence with reference serum values). the limited diagnostic accuracy for current dehydration by osmolality at tbw% of 46% (sensitivity 67%, specificity 62%) using the impedance output from mf - bia to calculate tbw% suggests that only 67 of every 100 people with current dehydration by serum osmolality will be positive using tbw% as the test, meaning that 33 of every 100 with current dehydration will be missed. similarly the specificity of 62% suggests that for every 100 people without current dehydration 62 will have a negative test but 38 will have a positive test2. this is a very high level of false positives and negatives, suggesting that mf - bia is not useful in diagnosing water - loss dehydration independently of other clinical or biochemical data. the test s positive (ppv) and negative (npv) predictive values3 as well as pre and post - test probabilities provide more information on the utility of tbw% at the 46% cut off point. the ppv of 33% (equivalent to the positive post - test probability of 33%) suggests that only 33% of those who are diagnosed as having current dehydration by mf - bia truly have current dehydration by serum osmolality. the npv of 87% is clearly better, meaning that 87% of those diagnosed as not having current dehydration are truly without current dehydration (and this is another way of stating the negative post - test probability of 13%). the positive likelihood ratio (lr) was 1.75 and negative likelihood ratio (lr) 0.544 suggesting that for a person positive for dehydration by this test the odds are 1.75 that dehydration is present compared to 1.00 for a person studies evaluating the utility of mf - bia in diagnosing dehydration in clinical settings are scarce. meta - analysis of studies in which tbw was estimated using bia and validated against the reference standard method, deuterium isotope dilution, found that mf - bia estimated tbw more closely to the reference standard (wmd 0.18l, 95% ci 1.62 to 1.98) than either single frequency (sf) bia (wmd 3.00l, 95% ci 1.43 to 4.57) or bioelectric impedance spectroscopy (wmd 2.80l, 95% ci 1.03 to 4.58). however, there are other indications in the literature that assessment of tbw may not be sufficiently accurate in conditions of change in hydration status and when body compartments are undergoing acute changes. this may be because changes in the ratio of intra- to extra - cellular water, and of acute changes in these compartments, also influence resistivity [2629 ]. this may mean that there are fundamental problems with mf - bia in assessing tbw and so in using mf - bia in predicting hydration status. our results suggesting that mf - bia is not a useful diagnostic tool are in broad agreement with those of olde rikkert 1997. they found that in dehydrated geriatric patients (n=53) the sensitivity of diagnosing dehydration using 100 khz mf - bia measurements was only 14% very poor sensitivity, and sensitivity was not improved when other frequencies were tested. the sensitivity and specificity of mf - bia were similar to that of the much simpler measure, tongue dryness, found in a recent australian study. tongue dryness is easier and quicker to assess, and does not need additional investment or electrical supplies, so would be a more appropriate measure to use in most situations than mf - bia if its diagnostic accuracy is similar. leaving the mathematics of diagnostic accuracy of mf - bia aside and observing data generated by mf - bia also suggested that mf - bia generated outcomes are not coherent with the diagnosis of dehydration. table 2 suggested no significant difference in mf - bia measures between hydrated, impending, and currently dehydrated groups.. cellular hydration status can change within minutes under the effects of stress, nutrients, hormones, and other factors. mf - bia does not appear to usefully reflect changes observed in serum osmolality or osmolarity or to sensitively identify the dehydrated state at the cellular level. haussinger suggested that while a well hydrated cell increases anabolic processes, a dehydrated cell shifts metabolism to catabolism, especially in muscle tissue. if recovery is to occur in a highly stressed patient after stroke, we want to ensure they are in an anabolic rather than a catabolic state. bhalla found that the 30% of their 167 stroke patients who had raised serum osmolality (> 296 mosm / kg) had increased odds of mortality at 3 months (or 2.4, 95%ci 1.0 t 5.9). kelly found that in their 102 acute ischaemic stroke patients raised serum osmolality (> 297 mosm / kg, in 24% of their patients) on day 9 following admission was associated with increased odds of venous thromboembolism (or 4.7, 95% ci 1.4 to 16.3). the maltron website states that the bioscan 920 - 2 multi - frequency analyser with its unique features is a rapid, non - invasive, inexpensive method for evaluating hydration and nutrition status it suggests applications in fluid retention and effects of hydration and dehydration. despite the maltron website reporting that it is quick, safe and easy and no assistance or technical knowledge is required (see www.maltronint.com/popup_pages/bioscan9202.htm, accessed 23/2/2012) we found the machine is not user friendly. without a keyboard, data entry and saving of data are slow and may result in errors and data loss. re - running a second measurement for the same participant requires re - entering all the same information again or the new test overwrites existing data. analysed data are not easily accessible to visual check without downloading the full data set, and there is no warning when unrealistic readings are registered. on - site readout of each variable for each participant was time consuming and unrealistic in an acute stroke unit. in assessing mf - bia we followed the guidelines of the european society of clinical nutrition and metabolism. the battery was charged between measurements, cables were of appropriate length, and the equipment calibrated for all participants reported in this paper. weight and height were measured by the investigator or by a health professional (not self - reported), mf - bia measurements were carried out at ambient temperature and manufacturer guidelines on positioning of electrodes were followed. before measurements our participants were in a supine position for least 10 min and during measurement they had no contact with any metal object (such as a bed frame). electrodes were attached to the non affected side to record measurements and to skin with no abrasions or deformation that may affect current conductance. none of our participants suffered from edema due to excessive hydration by iv / electrolyte infusion or very low albumin levels. one of the main limitations in a ward setting is the inability of researchers to completely control participants fasting or bladder voidance, but the investigator did ensure that participants had fasted for at least 2 hours before mf - bia measurements were taken and all participants were asked if they would like to void their bladder before measurements commenced. study strengths included the use of serum osmolality and calculated osmolarity as reference standards, a population with high levels of dehydration, and recording serum osmolality and other serum measures (sodium, potassium, glucose, urea) within 20 minutes of mf - bia measurements (enabling us to capture cellular hydration status as evaluated by mf - bia and its coherence with reference serum values). mf - bia does not appear appropriate for the diagnosis of water - loss dehydration after stroke. diagnostic accuracy is far too low to usefully diagnose current or impending dehydration at any selected cut - off point. however, separating assessment by sex, and using tbw as a percentage of lean body weight may warrant further investigation. | backgroundnon - invasive methods for detecting water - loss dehydration following acute stroke would be clinically useful. we evaluated the diagnostic accuracy of multi - frequency bioelectrical impedance analysis (mf - bia) against reference standards serum osmolality and osmolarity.material/methodspatients admitted to an acute stroke unit were recruited. blood samples for electrolytes and osmolality were taken within 20 minutes of mf - bia. total body water (tbw%), intracellular (icw%) and extracellular water (ecw%), as percentages of total body weight, were calculated by mf - bia equipment and from impedance measures using published equations for older people. these were compared to hydration status (based on serum osmolality and calculated osmolarity). the most promising receiver operating characteristics curves were plotted.results27 stroke patients were recruited (mean age 71.3, sd10.7). only a tbw% cut - off at 46% was consistent with current dehydration (serum osmolality > 300 mosm / kg) and tbw% at 47% impending dehydration (calculated osmolarity 295300 mosm / l) with sensitivity and specificity both > 60%. even here diagnostic accuracy of mf - bia was poor, a third of those with dehydration were wrongly classified as hydrated and a third classified as dehydrated were well hydrated. secondary analyses assessing diagnostic accuracy of tbw% for men and women separately, and using tbw as a percentage of lean body mass showed some promise, but did not provide diagnostically accurate measures across the population.conclusionsmf-bia appears ineffective at diagnosing water - loss dehydration after stroke and can not be recommended as a test for dehydration, but separating assessment by sex, and using tbw as a percentage of lean body weight may warrant further investigation. |
a computerized literature search for human and animal studies was performed in the pubmed and cochrane databases, till june 2012. in addition, a hand search was carried out in the following journals : international journal of oral and maxillofacial surgery, british journal of oral and maxillofacial surgery, journal of oral and maxillofacial surgery, journal of cranio - maxillofacial surgery, clinical implant dentistry and related research, clinical oral implants research, journal of oral implantology, the international journal of oral and maxillofacial implants, international journal of periodontics and restorative dentistry, journal of clinical periodontology, journal of periodontology, periodontology 2000, international journal of prosthodontics, journal of prosthetic dentistry, oral surgery oral medicine oral pathology. further, we checked the cochrane controlled trials register and the cochrane health group specialized register for publications on sinus floor augmentation. articles were searched from 1997 (the first known study of sinus lift surgery without the use of grafts) to june 2012. blood or venous blood or peripheral venous blood or gelatin sponge or resorbable device. the inclusion criteria for the articles to be included in this present systematic review were as follows : articles that showed evidence of atrophy of the maxilla in the premolar / molar area that required a sinus lift before implantation in case of human studiescase series in which bone graft or bone substitutes were not used in the maxillary sinus augmentationclinical trials in which bone graft or bone substitutes were not used in any one of the groupsarticles having information on either animals / humans in this areaarticles with either histological or radiographic evaluation for maxillary sinus floor augmentation. articles that showed evidence of atrophy of the maxilla in the premolar / molar area that required a sinus lift before implantation in case of human studies case series in which bone graft or bone substitutes were not used in the maxillary sinus augmentation clinical trials in which bone graft or bone substitutes were not used in any one of the groups articles having information on either animals / humans in this area articles with either histological or radiographic evaluation for maxillary sinus floor augmentation. articles having studies not done with lateral window approacharticles other than in english languagearticles having studies done with less than 6 months follow - up. articles having studies not done with lateral window approach articles other than in english language articles having studies done with less than 6 months follow - up. titles derived from this broad search were independently screened by two authors based on the inclusion criteria. abstracts of all titles agreed on by both authors were obtained and screened for meeting the inclusion criteria. if no abstract was available in the database, the abstract of the printed article was used. if the title and abstract did not provide sufficient information regarding the inclusion criteria, the full report was obtained as well. finally, the selection based on inclusion and exclusion criteria was made for the full - text articles. for this purpose, material and methods and results sections of these studies reasons for exclusion of articles from the present review have been tabulated in table 2. data was extracted using data extraction tables from the selected papers based on author(s), year of publication, study design, human study or animal study, sample size, inclusion and exclusion criteria, follow - up period, surgical technique, height of residual alveolar crest, material used for sinus floor augmentation, bone gain, method of examination of bone gain, sinus mucosa perforation, patient morbidity and disease transmission. the study design and basic data are presented in tables 3 and 4, while the summary of surgical procedures and variables of interest evaluated are given in tables 5 and 6. general information of selected (non - comparative) articles general information of selected (comparative) articles summary of surgical procedure and type of grafting material for selected non - comparative studies summary of surgical procedure and type of grafting material for selected comparative studies keywords were sinus augmentation or maxillary sinus augmentation or maxillary sinus floor augmentation and blood or venous blood or peripheral venous blood or gelatin sponge or fibrin rich block or platelet concentrates or space maintaining device or resorbable device. the inclusion criteria for the articles to be included in this present systematic review were as follows : articles that showed evidence of atrophy of the maxilla in the premolar / molar area that required a sinus lift before implantation in case of human studiescase series in which bone graft or bone substitutes were not used in the maxillary sinus augmentationclinical trials in which bone graft or bone substitutes were not used in any one of the groupsarticles having information on either animals / humans in this areaarticles with either histological or radiographic evaluation for maxillary sinus floor augmentation. articles that showed evidence of atrophy of the maxilla in the premolar / molar area that required a sinus lift before implantation in case of human studies case series in which bone graft or bone substitutes were not used in the maxillary sinus augmentation clinical trials in which bone graft or bone substitutes were not used in any one of the groups articles having information on either animals / humans in this area articles with either histological or radiographic evaluation for maxillary sinus floor augmentation. articles having studies not done with lateral window approacharticles other than in english languagearticles having studies done with less than 6 months follow - up. articles having studies not done with lateral window approach articles other than in english language articles having studies done with less than 6 months follow - up. titles derived from this broad search were independently screened by two authors based on the inclusion criteria. abstracts of all titles agreed on by both authors were obtained and screened for meeting the inclusion criteria. if no abstract was available in the database, the abstract of the printed article was used. if the title and abstract did not provide sufficient information regarding the inclusion criteria, the full report was obtained as well. finally, the selection based on inclusion and exclusion criteria was made for the full - text articles. for this purpose, material and methods and results sections of these studies were screened. reasons for exclusion of articles from the present review have been tabulated in table 2. data was extracted using data extraction tables from the selected papers based on author(s), year of publication, study design, human study or animal study, sample size, inclusion and exclusion criteria, follow - up period, surgical technique, height of residual alveolar crest, material used for sinus floor augmentation, bone gain, method of examination of bone gain, sinus mucosa perforation, patient morbidity and disease transmission. the study design and basic data are presented in tables 3 and 4, while the summary of surgical procedures and variables of interest evaluated are given in tables 5 and 6. general information of selected (non - comparative) articles general information of selected (comparative) articles summary of surgical procedure and type of grafting material for selected non - comparative studies summary of surgical procedure and type of grafting material for selected comparative studies among the 22 articles included in the review, most of them revealed the presence of bone formation in the maxillary sinus augmentation without bone grafts. following is a count of the number of articles based on the grafting material used : blood - 13, platelet rich fibrin - 2, space maintaining device - 4, platelet rich plasma - 1, fibrin rich block - 1, and gel sponge - 1. out of the 22 articles included, 16 were human studies and 6 were animal studies. though most of the articles reviewed showed evidence of bone formation, variation in levels of bone formation was noticed among different groups as well as among different studies. the primary objective of the present review was to assess the effectiveness of bone gain in the maxillary sinus floor augmentation without any bone grafts using lateral window technique. level of evidence of selected articles from the centre for evidence - based medicine, oxford in the present review, 13 studies were included to evaluate the effect of bone gain in the maxillary sinus floor augmentation with implant and blood alone using lateral window technique. among these, using blood as sole grafting material, presence of bone gain was shown in all cases. long - term human studies using blood as grafting material in sinus augmentation and with a minimum of 1 year follow - up also revealed the presence of bone gain in most of the cases. in the short - term human studies, though there was evidence of new bone formation, it required long - term follow - up to arrive at conclusions. maxillary sinus augmentation with blood showed no statistical difference in the amount of bone gain when compared to the autogenous grafts. thus this technique is considered to be cost - effective, less time - consuming and associated with lower morbidity since no bone harvesting is needed summary of published studies (1997 - 2011) of sinus lift with blood only is presented in table 8. summary of studies of sinus lift with blood only the maxillary sinus floor augmentation with platelet rich fibrin, platelet rich plasma, fibrin rich block, gel sponge, and space maintaining device also showed evidence of bone formation with varying levels of bone formation among different groups. all the 22 articles included in the review showed predictable amount of bone formation in the maxillary sinus augmentation. the membrane elevation technique without the use of grafting materials, as described in this paper, is not initially an easy technique as it may require adaptation from the more common technique using grafts where the bony window is prepared with a burr. however, with results comparable to the routine sinus lift techniques, the membrane elevation technique displayed possible advantages. however, the problems encountered such as sinus septae and membrane perforations are factors that need to be taken in to consideration. these must be taken into account during the surgical planning of the procedure and are best visualized by preoperative computed tomography evaluation. the premolar region is also the location of most septae in atrophic edentulous ridges and it has been shown that septae in dentate maxillae are of greater heights than in edentulous patients. while planning the surgery, however, they might prove to be helpful in achieving satisfactory primary stability for the implant when placed in these septae of the basal bone of the maxillary atrophied crest. the use of a wide bony window for access to the sinus mucosa is also important. the anatomical features like septae and a fragile mucosa may develop into lacerations of the sinus mucosa during the dissection, which requires careful and delicate surgical approach. it is evident from the data available in existing systematic reviews that there are reported cases of the sinus membrane perforation and it might be one of the reasons affecting the success rate of the procedure. suturing of the mucosa to the superior part of the bony window after extensive dissection has also been recommended, but not yet evaluated by randomized controlled trials. the articles included in this review show evidence of good survival rate when implant is placed either along with or after maxillary sinus augmentation without bone grafts. has shown that augmenting maxillary sinus using space maintaining device without bone grafts was associated with evidence of more bone formation when placed simultaneously along with maxillary sinus augmentation. the implant survival rate depends on various conditions such as quality of the bone, stage of implant installation, implant system, implant surface, implant length, implant diameter, prosthetic considerations, systemic health of the patient, and oral hygiene status. the unpublished and the raw data of few studies have not been included for interpretation. due to lack of studies available, the standardization of methodology was not considered, which might have led to difficulty in comparison of bone gain among different group of non - bone grafting materials. further, only articles that had minimum 6 months follow - up were included, for better observation of bone formation, randomized controlled trials with long - term follow - up would be required. variations have been observed in the study design adopted, methodology used, type of grafting material used, follow - up period and method of evaluation of bone gain among the different articles. selection and inclusion of randomized controlled trials with long - term follow - up and studies with standardization in study design, methodology, follow - up period and method of evaluation of bone gain are required to obtain a clear interpretation of the effectiveness of maxillary sinus augmentation without bone grafts. the membrane elevation technique without the use of grafting materials, as described in this paper, is not initially an easy technique as it may require adaptation from the more common technique using grafts where the bony window is prepared with a burr. however, with results comparable to the routine sinus lift techniques, the membrane elevation technique displayed possible advantages. however, the problems encountered such as sinus septae and membrane perforations are factors that need to be taken in to consideration. these must be taken into account during the surgical planning of the procedure and are best visualized by preoperative computed tomography evaluation. the premolar region is also the location of most septae in atrophic edentulous ridges and it has been shown that septae in dentate maxillae are of greater heights than in edentulous patients. while planning the surgery, however, they might prove to be helpful in achieving satisfactory primary stability for the implant when placed in these septae of the basal bone of the maxillary atrophied crest. the use of a wide bony window for access to the sinus mucosa is also important. the anatomical features like septae and a fragile mucosa may develop into lacerations of the sinus mucosa during the dissection, which requires careful and delicate surgical approach. it is evident from the data available in existing systematic reviews that there are reported cases of the sinus membrane perforation and it might be one of the reasons affecting the success rate of the procedure. suturing of the mucosa to the superior part of the bony window after extensive dissection has also been recommended, but not yet evaluated by randomized controlled trials. the articles included in this review show evidence of good survival rate when implant is placed either along with or after maxillary sinus augmentation without bone grafts. has shown that augmenting maxillary sinus using space maintaining device without bone grafts was associated with evidence of more bone formation when placed simultaneously along with maxillary sinus augmentation. the implant survival rate depends on various conditions such as quality of the bone, stage of implant installation, implant system, implant surface, implant length, implant diameter, prosthetic considerations, systemic health of the patient, and oral hygiene status. the unpublished and the raw data of few studies have not been included for interpretation. due to lack of studies available, the standardization of methodology was not considered, which might have led to difficulty in comparison of bone gain among different group of non - bone grafting materials. further, only articles that had minimum 6 months follow - up were included, for better observation of bone formation, randomized controlled trials with long - term follow - up would be required. variations have been observed in the study design adopted, methodology used, type of grafting material used, follow - up period and method of evaluation of bone gain among the different articles. selection and inclusion of randomized controlled trials with long - term follow - up and studies with standardization in study design, methodology, follow - up period and method of evaluation of bone gain are required to obtain a clear interpretation of the effectiveness of maxillary sinus augmentation without bone grafts. within the limits of the articles and data available, maxillary sinus augmentation without bone graft might be considered effective in predictable bone formation. | objective : the objective of the present review was to determine the effectiveness of maxillary sinus floor augmentation without bone grafts using lateral window technique.materials and methods : pubmed and cochrane databases were searched for relevant articles. we also included articles by hand search until june 2012. the analysis included both human and animal studies which satisfied the following criteria : minimum of 6 months follow - up, no use of bone grafts, and lateral window approach to the sinus.results:we included 22 articles in the review. a descriptive analysis of the constructed evidence tables indicated that there is evidence of predictable a mount of bone formation in the maxillary sinus augmentation without the use of bone grafts.conclusion:within the limits of the articles and data available, maxillary sinus augmentation without bone graft might be considered effective inpredictable bone formation. |
while affiliative experiences may be rewarding, social situations and hierarchies may also be associated with negative affects such as anxiety. given the centrality of affiliative and social concerns in human life, the high prevalence of social anxiety is not surprising. furthermore, it is not surprising that social anxiety disorder (sad) is accompanied by significant distress and functional impairment. behavioral inhibition, characterized by socially fearful and avoidant behavior, is both a feature of sad and a key predictor for the development of this condition. both in daily life and in experimental work, such social fear and avoidance in sad manifests in terms of blushing, as well as avoidant or submissive responses to the eye gazes of others. blushing and gaze aversion in sad are arguably a pathological manifestation of an evolutionarily evolved submissive response that appears in primate dominance encounters. while rodents dominate by means of aggression, in primates subordinates avert their gaze when challenged by the threat stare of the dominant animal, so allowing the social hierarchy to be regulated nonaggressively. in sad, however, such social adaptations seem to have gone awry and socially fearful, avoidant, and submissive behaviors are generalized to all of human social interaction. animal research has established a pivotal role for the neuropeptide oxytocin (oxt) and the steroid testosterone (t) in the development and adaptive preservation of social hierarchies. such work, as well as placebo - controlled studies with single administrations of oxt and t in humans, leads to the hypothesis that imbalances in oxt and t systems may contribute to the pathogenesis of sad. in many species, including humans, the peptide hormone oxt and the steroid hormone t play a key role in the development and execution of social - emotional behavior, both in males and in females. depending on the relevant social context and environment, these social hormones act via, and interact with, all the main neurotransmitter systems (that is, the serotoninergic, noradrenergic, and dopaminergic systems). the other key social hormone systems, the estrogen and the vasopressin systems, depend critically on t, as the most important estrogen, estradiol, is a metabolite of t, and vasopressin depends on t for its gene expression. since oxt depends on estradiol for its gene expression, all of our social behavior is ultimately- based upon t, a hormone that is ironically associated with antisocial behavior in some folk psychologies. although oxt and t possess opposite behavioral properties, for example in the domain of cognitive empathy, they are certainly not antagonistic hormones in their effects on brain and behavior. on the contrary, the oxt and t systems are intrinsically intertwined, jointly critical for the execution of sexual behavior, and have important and seemingly complementary anxiolytic / fear - reducing properties. oxt and t act, directly or indirectly, via other hormone or neurotransmitter systems, on all social - affective brain systems including the orbitofrontal cortex (ofc), anterior cingulate cortex (acq, amygdala, ventral striatum, hypothalamus, and brain stem. however, depending on biological predisposition, early adversity (for example, abuse or neglect) can disorganize the expression of social - emotional behaviors by creating imbalances in hormonal systems, and especially in the oxt and t systems. these hormonal imbalances have negative effects on the social - emotional brain and can produce socially fearful, avoidant, and submissive behaviors, with insensitivity for social rewards. early - life social experiences shape the expression of adult social behaviors via neuropeptide and steroid systems, and especially the oxt and t systems, which among others organize the expression of fear and aggression, and related approach and avoidance behaviors. notably, changes in the production and secretion of oxt and t in response to social events, and changes in the social brain 's sensitivity to these hormones, are fundamental mechanisms by which social experience affects later social behaviors. furthermore, the social - emotional behaviors governed by the oxt and t systems seem to play a major role in sad. there is some evidence that oxt and t levels measured in plasma or saliva are lowered in sad. however, saliva and plasma measures only coarsely- reflect brain oxt and t, as oxt does not cross the bloodbrain barrier, and t is produced not only by the gonads and adrenals, but also is a neurosteroid in the brain that is not measurable peripherally. what makes oxt and t of specific interest to sad are placebo - controlled oxt and t administration studies in humans, which suggest that these hormones can alleviate the core behavioral hallmarks of sad (social tearfulness, avoidance, submissiveness), and increase sensitivity to social rewards, as described in more detail below. the robust evidence for acute behavioral plasticity in the oxt and t systems, with transient development of new social behaviors in adulthood in response to single administrations of oxt and t, potentially opens up significant therapeutic opportunities, particularly in light of one of the important scientific discoveries of recent decades, that of adult neuroplasticity. adult neuroplasticity refers to structural changes in brain regions and rewiring of brain pathways in adulthood, and social experiences. oxt and t not only develop and execute our social - emotional behavior, but they also are key players in adult neuroplasticity. importantly, oxt and t are able to open up new developmental windows in adulthood, and on these windows social experiences (and thus psychosocial interventions) can potentially act to bring lasting change (figure 1). in rodent research, the anxiolytic or (social) fearreducing properties of oxt and t have been well established. t elevations in rats increase social exploration, while decreasing anxiety, punishment, and avoidance. compared with intact male rats and/or gonad ectomized rats with replacement hormone, gonadectomized rats show more anxiety, fear, and freezing behavior on a large variety of tasks. importantly, anxiolytic / fear - reducing effects of oxt are often observed in social situations in rodents. notably, after being defeated in dominance interactions, rats display social avoidance, but not after administration of oxt. oxt acts on a localized population of oxt receptors in the amygdala to reduce fear behaviors. oxt also modulates activity within the hypothalamic - pituitary - adrenal (hpa) axis, augmenting hpa - axis activation immediately after stress exposure. this arguably facilitates system adaptation, and subsequently enhances the suppression of the iipa axis, which helps in the re - establishment of system homeostasis. t also has multiple controls over the iipa axis ; t can inhibit the hpa axis at the hypothalamic, pituitary, and adrenal levels. thus, oxt and t not only have numerous anxiolytic / fear reducing actions, especially in the social domain, but also have powerful control over hpa function and activity, particularly during stress. the evidence for anxiolytic and fear - reducing effects of oxt and t in humans is based upon recent studies with single intranasal administrations of oxt and single sublingual administrations of t. using a placebo - controlled single testosterone administration method developed for human females with an uniquely established quantity and time course of effect (the tuiten method), the fear and stress - reducing properties of the steroid hormone testosterone in humans have been demonstrated repeatedly by van honk and colleagues. early human data on the relation between testosterone, fear, and anxiety predominantly involved questionnaires that index the conscious appraisal of anxious mood. however, testosterone does not acutely act on such emotions, but only on genuine fear behaviors. indeed, in the absence of any effects on anxiety, van honk showed significant reductions in vigilant responses to masked facial fear after testosterone administration. hermans measured baseline startle and fear - potentiated startle in a threat - of - shock paradigm, and showed that testosterone administration acutely reduced the fearpotentiated startle, but did not affect baseline startle. finally, hermans showed reductions in skin conductance and startle modulation in response to stress - inducing negative and threatening pictures in anxiety - prone participants. angry facial expressions are thought to have evolved in primates with a key function being social threat signaling in dominance encounters. in face - to - face challenges between primates, high levels of testosterone relate to social dominance in numerous species including humans. indeed, using an adapted emotional stroop task it was shown that vigilant responses (enduring gazes) to angry facial expressions are positively related to testosterone levels in both males and females. in these findings however, with a placebo controlled testosterone administration design (the tuiten method), it was shown that t induces cardiac acceleration to exclusively angry faces in healthy women, indicating that the hormone plays a causal role in encourages dominance behavior. furthermore, using the same method and testing subjects in a social approach - avoidance task, enter showed reductions in avoidant responses to facial anger, suggesting that t counteracts submissive responses of eye and gaze aversion to facial anger. this finding is relevant to sad, because socially anxious subjects are strongly avoidant to facial anger in the same social approach - avoidance task. terburg developed an eye - tracking paradigm to measure true gaze aversion and staring endurance in unconscious face - to - face confrontations, with backwardly masked angry faces. backward masking is a phenomenon wherein the presentation of a target stimulus (here the angry face) is immediately followed (in this case 30 milliseconds), by a masking stimulus with the same visual information but scrambled, which results in a failure to consciously perceive the target stimulus. with this paradigm it was shown that on the dominance - submissive spectrum, relatively high dominance traits predict a prolonged gaze to masked facial anger (enduring gaze), while relatively low dominance traits predict gaze aversion. crucially, terburg next used acute t administration and showed that individual response patterns shifted from gaze aversion to staring endurance. t thus seems to lead to social dominance in humans (and likely also in other mammals and in reptiles) implicitly and nonconsciously. on the other hand, with their social approach - avoidance task, which operates at more explicit, conscious levels of processing, radke and coworkers not only showed that avoidant response to angry facial expressions are positively related to social anxiety levels, but also that t reduced this socially avoidant behavior. taken together, corresponding to the effects on baseline anxiety and cue - specific fear, t and oxt seem to influence dominance - submissive behaviors at different levels of processing. t operates on the most implicit nonconscious level of processing while oxt also acts on automatic behaviors but only at higher, more explicit, levels of information processing. furthermore, it has repeatedly been shown that oxt improves or increases the processing of happy facial expressions. these data are consistent with findings that intranasal oxt generally leads to increased attention to the eyes of others both in experimental and realistic conditions. interestingly, t and oxt may act in opposite ways in the case of facial happiness, as the happy face is also an appeasement signal. t may lead to social approaches in the context of competition eg, for dominance, but not in other situations. in contrast, oxt seems to make social interactions in general more rewarding, and the hormone therefore enhances the processing of facial happiness. in sum, oxt may increase attention to, and memory for, positive facial expressions, because the hormone increases the rewarding properties of social interactions. t especially induces vigilant attention and responsivity to angry facial expressions, because the angry face functions as a dominance signal in face - to - face interactions, thus social dominance is at stake and the hormone is on its guard. overall, oxt reduces background anxiety, and improves the recognition of, and attention to, positive facial expression. oxt also increases attention to the eye region of the face, a sign of interest in social others, and of the willingness to socialize. thus, the social peptide oxt seem to hold properties that make social interaction more rewarding. the social steroid t on the other hand reduces cue - specific and social fear, and submissiveness and facilitates vigilance and social approach but exclusively in competitive conditions : to compete, approach and defend status in social interactions. anxiety, social fear and avoidance, and impaired social reward processing all are behavioral hallmarks of sad, but the specific actions of oxt and t may suggest a personalized approach to their use in therapeutic interventions. that is, individuals suffering from sad and a lack of motivation to socialize may show a response to oxt, while socially fearful, submissive sad subjects who are eager to interact may show a response to t. importantly, as is clear from the earlier discussion, a range of validated behavioral, neuropsychological, and psychophysiological paradigms are available to assess putative behavioral differences in sad. it is important to note that the effects of oxt and t may depend upon various personal and situational factors. this represents another potential opportunity ; that is, for employing oxt and t administration in synergy with exposure to social experiences as part of an innovative combined treatment strategy in sad. to date, however, research with intranasal oxt in sad is still scarce, and published research in sad with t administration is nonexistent. one trial with intranasal oxt adjunctive to exposure therapy in sad showed improvement in the mental representation of the self. further, two pharmacological neuroimaging studies in patients with sad receiving intranasal oxt and placebo showed attenuation of amygdala activity to fear, and the modulation of medial frontal hyperactivity to sad faces. taken together these preliminary data are promising and provide a foundation for additional research on oxt administration in sad. the modern behavioral and psychophysiological paradigms tapping into social fearfulness, social avoidance, submissiveness, and social reward processing discussed above may be particularly useful in further investigations of the assessment and treatment of sad. research on oxt and t has suggested that these hormones alter such paradigms in overlapping but distinct ways. future research on pathogenesis may usefully focus on identifying subtypes of sad which respond differentially to oxt and t administration. ultimately, a better understanding of oxt and t, as well as the pathogenesis of sad may lead to interventional research which optimally integrates psychotherapy with adjunctive hormone administration. | social anxiety disorder (sad) is a highly prevalent and disabling disorder with key behavioral traits of social fearfulness, social avoidance, and submissiveness. here we argue that hormonal systems play a key role in mediating social anxiety, and so may be important in sad. hormonal alterations, often established early in development through the interaction between biological and psychological factors (eg, genetic predisposition x early trauma), predispose to socially fearful, avoidant, and submissive behavior. however, whereas gene variants and histories of trauma persist, hormonal systems can be remodeled over the course of life. hormones play a key role during the periods of all sensitive developmental windows (ie, prenatal, neonatal, puberty, aging), and are capable of opening up new developmental windows in adulthood. indeed, the developmental plasticity of our social brain, and thus of social behavior in adulthood, critically depends on steroid hormones such as testosterone and peptide hormones such as oxytocin. these steroid and peptide hormones in interaction with social experiences may have potential for reprogramming the socially anxious brain. certainly, single administrations of oxytocin and testosterone in humans reduce socially fearful, avoidant, and submissive behavior. such work may ultimately lead to new approaches to the treatment of sad. |
nonalcoholic fatty liver disease (nafld) has become a significant cause of chronic liver disease with staggering occurrence in the usa and worldwide. nafld disease prevalence studies estimate anywhere between 2.8% and 38.5% of the us general population has nafld [26 ]. epidemiological studies indicate nafld is pathophysiologically linked to metabolic syndrome as it is associated with obesity, hypertension, dyslipidemia, and insulin resistance [7, 8 ]. nafld is a term used to label a spectrum of liver diseases ranging from early stage fatty liver (steatosis) to advanced cirrhosis of the liver and hepatocellular carcinoma. this disease is aptly named as it occurs in individuals who consume little to no alcohol and the nafld pathology closely resembles that of a diseased liver attributable to alcohol abuse. nafld is believed to originate with the accumulation of fatty acids within the liver as a result of insulin resistance. consequently increased membrane lipid peroxidation and oxidative stress within the cells of the liver result in inflammation and increased deposition of extracellular matrix (ecm) proteins, that is, fibrosis. excessive fibrosis leads to atypical hepatic arrangement and subsequent scarring. as tissue scarring develops there genetic factors contribute to an individual 's predisposition to the development and progression of nafld. the development of hepatic fibrosis is regulated primarily by hepatic stellate cells (hscs), which synthesize ecm proteins. in a quiescent state, hscs store vitamin a as retinol esters and make up roughly one - third of nonparenchymal cells of the liver. hscs are activated by injured hepatocytes and stimulated resident macrophages known as kupffer cells (kc) [16, 17 ]. kc play a significant role in immune surveillance and production of cytokines such as tumor necrosis factor (tnf-), interleukin-1 (il-1), and interleukin-6 (il-6). interestingly kc have been shown to be essential for liver regeneration after partial hepatectomy of the liver via cytokine production [20, 21 ]. kc and their immune response to various toxic insults play a role in disease progression and hepatic homeostasis. the inflammasome is a multimeric protein complex that is assembled and activated upon the detection of cellular infection or cell stress. damage - associated molecular patterns (damp) and pathogen - associated molecular patterns (pamp) bind to pattern - recognition receptors inducing an intracellular signaling cascade. these events lead to oligomerization of inflammasome components, activation of intracellular cysteine protease caspase-1, and the subsequent cleavage and maturation of proinflammatory cytokines il-1 and il-18 [2427 ]. the nlrc4 inflammasome detects the cytosolic presence of bacterial flagellin during infection using nlr family, apoptosis inhibitory protein 5 (naip5), and nlr family, card domain containing 4 (nlrc4) to form a heterooligomeric inflammasome structure [28, 29 ]. several activated nlrc4s then form an inflammasome complex by which nlrc4 uses an n - terminal caspase activating and recruitment domain (card) to interact with the card of pro - caspase-1, leading to cleavage and activation of caspase-1. the nlrc4 inflammasome may associate with caspase-1 independent of adaptor protein apoptosis - associated speck - like protein containing a caspase recruitment domain (asc) which itself contains a card. identification of genetic factors contributing to the pathogenesis of nafld facilitates the potential to target susceptible individuals for interventional strategies to ameliorate nafld progression. genome - wide association studies (gwas) are an unbiased tool for the identification of gene variants associated with genetic traits. unfortunately, gwas examining phenotypes relevant to nafld are lacking a 2015 gwas found the nlrc4 inflammasome was involved in il-18 production in patients with acute coronary syndromes. association studies such as these should be used in conjunction with hypothesis - driven investigative studies to identify key genetic regulators of nafld. we have previously shown allelic differences of the nlrc4 gene between inbred genetic mouse strains c57bl/6j (b6) and a / j modulate the development and/or resolution of hepatic fibrosis, a critical stage of nafld development. identifying the molecular mechanisms by which nlrc4 modulates liver fibrosis is essential to fully understand the complex dynamics of liver disease origin and development. using models of hepatotoxin - induced liver injury and liver regeneration after partial hepatectomy all procedures involving animals were approved by the case western reserve university institutional animal care and use committee. b6 and a / j mice were obtained from jackson laboratories and maintained at case western reserve university for over 10 generations. mice were housed in a microisolator environment on a 12 hr : 12 hr light / dark cycle. all mice were weaned at 3 - 4 weeks of age and maintained on labdiet # 5010 autoclavable rodent chow (labdiet, richmond, in) with food and water provided ad libitum. for both acute and chronic ccl4 studies, ccl4 (sigma - aldrich, st. all control mice receiving olive oil were sacrificed 48 hours after injection. for chronic ccl4 administration, mice were given two injections weekly (tuesday / friday) for 5 weeks. mice were gradually increased in dose of ccl4 over the first three injections (first, 0.25 l / g body weight ; second, 0.5 l / g body weight ; third and subsequent doses, 1 l / g body weight). both ccl4 and olive oil treated mice were sacrificed 72 hours after the final injection. mice were allowed access to food and water ad libitum for the duration of the study. blood plasma and liver tissue were collected and frozen until further processing. using custom designed primers that span the 1000 bp region preceding the start site of transcription for nlrc4, we isolated the nlrc4 promoter by polymerase chain reaction (pcr). each 1 kb promoter was cloned into a psc - a - amp / kan cloning vector using strataclone pcr cloning kit (agilent technologies, santa clara, ca) and then subcloned into the luciferase plasmid vector pgl4.10-luc2 using quicklink dna ligation kit (sigma - aldrich, st. the expression constructs were cotransfected with either an overexpression vector for cdx-1 driven by cytomegalovirus (cmv) promoter or a control vector containing only the cmv promoter (open biosystems products, huntsville, al) into murine macrophage cell line raw 264.7 using fugene hd transfection reagent (promega, madison, wi). cells were cultured using atcc - formulated dulbecco 's modified eagle 's medium (life technologies, grand island, ny) supplemented with 10% fetal bovine serum (life technologies, grand island, ny). dual - glo luciferase assay system (promega, madison, wi) activating firefly (photinus pyralis) and renilla (renilla reniformis) luciferases was used to control for transfection efficiency. luminescent signal was measured by plate - reading illuminometer (molecular devices, sunnyvale, ca) and activity was calculated according to the manufacturer 's protocol. chip was conducted on a cultured murine macrophage cell line raw 264.7 as previously described. the cells were cross - linked with 11% buffered formaldehyde solution and sonicated into dna fragments. dna fragments were selected using a cdx-1 antibody (abcam, cambridge, ma) conjugated with magnetic beads and purified. the fragments were analyzed by pcr (life technologies, grand island, ny). custom designed primers (idt, coralville, ia) were used to detect potential transcription factor cdx-1 binding sites (supplemental table 1 in supplementary material available online at http://dx.doi.org/10.1155/2015/909827). anti - gfp antibody (abcam, cambridge, ma) served as an antibody negative control. primers for a negative control locus and a nontemplate control were used during rt - pcr. 1216-week - old male b6 and congenic 17c-6 mice were anesthetized and their hind legs harvested from the pelvis keeping the femur bone intact. the surrounding skin and muscle tissues were removed revealing femur, tibia, and fibula bones. in an aseptic environment the femur and tibia bones were cut at each end and the lumen was flushed with culture media. cells were cultured in macrophage differentiation medium containing 10 ng / ml macrophage colony - stimulating factor (m - csf) at 37c in an air atmosphere with 5% co2 for 710 days to differentiate macrophage progenitor cells into mature macrophages as described [38, 39 ]. once bmdm were fully differentiated they were exposed to bacterial lps (100 ng / ml) for 2, 4, and 8 hours at 37c in an air atmosphere with 5% co2. subcellular fractionation of bmdm cells was prepared as previously described and either rna or protein was isolated. to test the role of caspase-1, we used 250 m selective irreversible caspase-1 inhibitor, acetyl - tyr - val - ala - asp - chloromethylketone (ac - yvad - cmk) (sigma - aldrich, st. the bmdm were isolated and incubated with the caspase-1 inhibitor yvad (50 m) for 0.5 hours. the bmdm were then stimulated with lps (200 ng / ml) for 4 hours while still exposed to the inhibitor and then treated with 1 mm atp for an additional 0.5 hours. after inhibitor incubation, lps priming, and atp incubation the cell - free media sample was collected and cells were washed twice with cold 1x pbs and then processed for analysis of protein and gene expression. lipofectin reagent was purchased from thermo fisher (grand island, ny) and used according to the manufacturer 's instructions. briefly, lipofectin reagent was prepared and incubated with bovine serum albumin as a control (500 ng / ml) for 24 hours or flagellin (500 ng / ml) for 24 hours. at the conclusion of the experiment cell media were collected and cells were lysed and the cell supernatant was collected. il-1 was measured by elisa (biolegend, san diego, ca) according to manufacturer 's instructions. 1216-week - old male b6 and congenic 17c-6 mice underwent two - thirds partial hepatectomy surgery performed by case western reserve university mouse metabolic phenotyping center (u24-dk76174) as previously described. the weight of resected liver for each mouse was recorded at the time of surgery. we found that resection of the median and left lateral lobes resulted in an average removal of 55% of total liver tissue. the animals recovered for 2-, 4-, 8-, 12-, 36-, and 168-hour (7 days) time periods and then were euthanized. blood plasma was taken and the remaining liver tissue was removed, weighed, and frozen. the animal survival rate was > 95% at all time points with no statistical difference between genotypes. percent liver regeneration was calculated by dividing the weight of the liver at the time of sacrifice by the initial liver weight of animal and multiplying by 100. the initial liver weight was obtained by assuming the resected liver weight was 55% of original liver mass [42, 43 ] : % liver regeneration = (a / b) 100, where a is liver weight at sacrifice, b is estimated liver weight before ph, b = (resected liver during ph)/0.55. % liver regeneration = (a / b) 100, where a is liver weight at sacrifice, b is estimated liver weight before ph, b = (resected liver during ph)/0.55. total rna from 30 mg of liver tissue was isolated from b6 and 17c-6 mice after 2/3 partial hepatectomy using nucleospin rna kit (macherey - nagel, bethlehem, pa). cdna was synthesized from 500 ng total rna using random hexamer primers and mmtv reverse transcriptase (applied biosystems, foster city, ca). real - time qpcr analysis was performed using bullseye evagreen sybr qpcr reagent (midsci, st. louis, mo) on a chromo4 cycler (mj research / bio - rad, hercules, ca). primer sequences were custom designed using primer3plus website version 2.3.6 (http://www.bioinformatics.nl/primer3plus/) (supplemental table 2). the ct value is converted to fold difference compared to endogenous housekeeping control by raising two to the ct [35, 44 ]. the levels of alt in plasma of b6 and 17c-6 mice after 2/3 partial hepatectomy were measured using a commercially available enzymatic assay kit (sekisui diagnostics, lexington, ma) as per manufacturer 's directions. whole cell protein isolation from b6 and 17c-6 mice after 2/3 partial hepatectomy was performed as previously described. polyvinyl difluoride membranes were incubated with an antibody for cyclin d1 (1 : 1,000 ; santa cruz biotechnology, dallas, tx), for stat3 and phosphorylated stat3 (tyr) (1 : 1,000 ; santa cruz biotechnology, dallas, tx). immunoreactive proteins were measured by scanning densitometry (un - scan - it software, orem, ut). western blots were normalized for loading differences using heat shock cognate protein 70 (hsc-70) (1 : 16,000 ; santa cruz biotechnology, dallas, tx) as previously described. blood plasma isolated from b6 and 17c-6 mice after 2/3 partial hepatectomy was used to measure il-1 (biolegend, san diego, ca), il-18 (ebioscience, san diego, ca), and il-6 (ebioscience, san diego, ca) by elisa according to manufacturer 's directions. the il-1 (biolegend, san diego, ca) elisa was also used for bmdm cell lysis supernatant discussed above. b6 and 17c-6 mice that had undergone 2/3 partial hepatectomy were intraperitoneally injected (ip) with bromodeoxyuridine (brdu) labeling reagent (life technologies, grand island, ny) 2 hours before sacrifice. freshly dissected liver was fixed in 10% neutral buffered formalin solution containing 4% formaldehyde for 48 hours and then embedded in paraffin and sectioned to 5 m and slide mounted. sections were deparaffinized, rehydrated using a series of graded alcohol solutions to 80%, and then trypsin digested. brdu staining procedure was done using brdu staining kit (life technologies, grand island, ny) according to kit manufacturer 's recommended instructions. images were obtained using a leica dm6000 upright microscope (case western reserve university imaging core facility, department of genetics and genome sciences, case western reserve university) and velocity acquisition software (perkinelmer, waltham, ma). four high - powered zones representative of slides were used to calculate positive brdu incorporation. the statistical differences reported are means standard error of the mean (sem). statistics were calculated by unpaired student 's t - test or by two - way anova with bonferroni correction for multiple testing using graphpad prism 5.0 software (graphpad, san diego, ca). gene sequencing analysis of the b6 and a / j nlrc4 promoter revealed a snp 331 bp upstream of the transcriptional start site (figure 1). the a / j promoter had a single thymine deletion at this site (rs74459439-t). transcription factor binding site analysis software (tfsearch internet - based tool) associated the location of this polymorphism as having a high likelihood for transcription factor cdx-1 binding (tfsearch score of 92.1). to definitively establish cdx-1 binding to the nlrc4 promoter, chromatin immunoprecipitation (chip) analysis was performed on raw 264.7 cells (b6 allele of the nlrc4 promoter) using primer sets spanning the snp, rs74459439-t. chromatin immunoprecipitation (chip) analysis revealed transcription factor cdx-1 binds to the mouse nlrc4 promoter at 3 potential cdx-1 binding sites but not at the region of the snp (rs74459439-t) (figure 2, supplemental figure 1). to investigate whether this snp results in nlrc4 expression differences we transfected pgl4.10 [luc2 ] luciferase vectors with the 1000-nucleotide promoter sequence from either the b6 or a / j allele, encompassing this snp. these constructs were independently cotransfected with an overexpression vector for transcription factor cdx-1 driven by cmv promoter or the cmv plasmid alone for control into murine raw 264.7 cells (figure 3). both the b6 and a / j nlrc4 promoter constructs had basal expression of luciferase activity and the luciferase activity was increased when cdx-1 was overexpressed. more importantly, the a / j allele shows a statistically higher luciferase activity than b6 with cdx-1 overexpression (figure 3). these results indicate not only that cdx-1 is playing a role in nlrc4 gene expression with murine macrophages, but also that there is a difference in its gene regulatory capacity contingent on the snp (rs74459439-t) version it contains. previously, deep sequencing analysis of the b6 and a / j mouse 17th chromosome revealed a snp (rs29502769) within exon 5 of nlrc4 resulting in the missense mutation i756v. we identified a new snp (rs74459439-t) in the promoter of nlrc4 (figure 1). to determine the effect these snps have on nlrc4 inflammasome activity, we measured the end product of the nlrc4 inflammasome, il-1. bmdm were isolated, differentiated, and cultured. the bmdm were stimulated with bacterial lipopolysaccharide (lps) (100 ng / ml) for 2, 4, and 8 hours. the 17c-6 bmdm containing the a / j allele of nlrc4 have increased cellular il-1 compared to b6. when 17c-6 bmdm are exposed to lps (200 ng / ml) for 24 hours they secrete equal amounts of il-1 into the media as b6 bmdm (figure 4(b)). looking inside the bmdm cell using the cell lysis supernatant after lps for 24 hours, the b6 and 17c-6 bmdm have equivalent concentrations (figure 4(c)). when stimulated with atp, il-1 secretion was comparable between b6 and 17c-6 demonstrating there are likely no differences in il-1 secretion biomechanics. it is possible that the nlrc4 inflammasome plays a role in inflammatory activity independent of normal inflammasome activation and subsequent caspase-1 cleavage / activation [46, 47 ]. in fact, the data in figures 4(d) and 4(e) indicate that nlrc4 may have a caspase-1 independent maturation and secretion of il-1. using a selective, irreversible inhibitor of caspase-1, ac - yvad - cmk, we observed a measurable increase in il-1 secretion into the cell media in bmdm derived from 17c-6 mice when compared to b6 mice. when caspase-1 was pretreated with its inhibitor, any subsequent il-1 maturation and secretion would be derived in a non - caspase-1 mediated fashion. mice with the a / j allele of nlrc4 are resistant to hepatotoxin ccl4-induced liver fibrosis, therefore, we hypothesize that nlrc4 may modulate the development of fibrosis. to determine if the function of nlrc4 impacts this process, we measured il-1 in b6 and 17c-6 mice after chronic exposure (5 weeks) to ccl4. we found that b6 mice have roughly a 3.3-fold increase in plasma il-1 after chronic ccl4 when compared to b6 olive oil injected control. congenic strain 17c-6, which has the a / j allele for nlrc4, had 1.3-fold increase over 17c-6 olive oil control. in addition, congenic 17c-6 mice had increased il-1 compared to b6 mice both for olive oil control (12.9-fold) and after chronic ccl4 exposure (5.2-fold) (figure 5(a)). thus, 17c-6 mice have constitutive activation of the nlrc4 inflammasome resulting in an increase of il-1 maturation and secretion into the plasma. to determine if the constitutive release of il-1 requires the stimulation with nlrc4 ligand, flagellin, we analyzed differentiated bmdm treated with bovine serum albumin (bsa) as a control (500 ng / ml) or flagellin (500 ng / ml) for 24 hours ; then the macrophages were stimulated with atp (5 mm) for 0.5 hours (figure 5(b)). at the conclusion of the experiment cell culture media there were no differences in the secretion of il-1 with flagellin suggesting that a noncanonical pathway is contributing to the increase of il-1. to fully test the livers ability to proliferate after injury a 2/3ph was performed on b6 and 17c-6 mice. time points chosen after 2/3ph were 2, 4, 8, 12, 36, and 168 hours after surgery. b6 and 17c-6 mice euthanized immediately after sham surgery showed 55% of original liver mass was remaining ; thus this is the starting point of percent original mass (figure 6). by 2 hours 17c-6 the divergence between the two groups becomes significant at 36 hours after surgery with 17c-6 animals averaging 87% original liver mass, while b6 animals average 57% of original liver mass (figure 6). both genotypes fully restored their initial liver mass by 7 days (168 hours) after 2/3ph surgery. it has been well established that tnf-, il-1, and il-6 activate transcription factors after hepatectomy. these include the nf-b signaling (phf / nf-b) and signal transducer and activator of transcription 3 (stat3) that are responsible for stimulation of the primary growth response or immediate - early genes and are rapidly activated after partial hepatectomy. nf-b signaling, phf / nf-b, is induced within 30 minutes after partial hepatectomy but quickly lost by 1 hour. stat3 induction is observed within 30 minutes after surgery and peaks between 3 to 5 hours after surgery, extending beyond the immediate - early time period. to determine if the key players in the primary growth response were altered we measured gene expression in livers from sham surgery b6 and 17c-6 mice. the mrna levels from these livers were quantified for il-1, interleukin-18 (il-18), tnf-, nf - kappa - b inhibitor- (ib), il-6, stat3, fbj osteosarcoma oncogene (c - fos), myelocytomatosis oncogene (c - myc), c - reactive protein (crp), and cyclin d1 (ccnd1) (figure 7(a)). we found elevated mrna levels in livers from 17c-6 mice compared to b6 mice for all of the genes measured. to determine if stat-3 protein levels varied after partial hepatectomy, we measured stat-3 phosphorylation by western blot analysis. we found increased stat-3 phosphorylation at 4 hours after 2/3 partial hepatectomy in 17c-6 mice compared to b6 mice (figure 7(b)). thus the livers of 17c-6 mice are primed for primary growth response in liver regeneration. to test their liver regeneration capacity we measured cyclin d1 protein content in the liver of b6 and 17c-6 mice after 2/3ph. the amount of cyclin d1 is variable over time with a decrease in cyclin d1 at 8 hours, which was shared by both genotypes. however, the 17c-6 mice had significantly greater cyclin d1 levels compared to b6 mice at sham and 4-, 12-, and 36-hour time points (figure 8). cyclin d1 is a prominent regulator of the cell cycle at the g1/s phase transition and an indicator of cell mitosis. to quantitatively measure the proliferation of cells within the liver at various time points following 2/3ph, we measured brdu incorporation into the newly synthesized dna of replicating cells. the images obtained from microscopy were quantified as percent positive area for brdu staining using imagej software (figure 9). this data showed increased positive staining of cells for brdu for 17c-6 mice over b6 mice beginning at the 8-hour time point and continuing to 12- and 36-hour time points. 17c-6 mice had increased positive staining for brdu between 4 and 36 hours after surgery. this is consistent with data in figures 6 and 8 as they indicate the majority of liver mass regeneration in the b6 mouse occurs between the 36 hours and 7 days after 2/3ph. the 17c-6 mice have markedly higher hepatic cell proliferation 8 hours after 2/3 partial hepatectomy, lasting at least until 36 hours after surgery when compared to b6 mice. this supports that 17c-6 mice have the capability for an early cell proliferative response during the recovery after 2/3ph (figure 9). to determine if the altered nlrc4 inflammasome activity impacts circulating inflammatory cytokine levels during liver regeneration, we measured cytokine il-18 in the plasma. plasma il-18 levels were unchanged after 2/3ph in both b6 and 17c-6 except at the sham and 36-hour time points. at sham, 17c-6 mice have a 2-fold increase over b6 mice and at 36 hours 17c-6 have nearly a 5-fold increase over b6 (figure 10(a)). il-18 is a potent inducer of il-6 in murine peritoneal macrophages and the role of il-6 in liver regeneration is well established. plasma il-6 levels were statistically increased in the sham surgery 17c-6 mice compared to b6 mice. il-6 plasma concentration increased in both b6 and 17c-6 at 2 hours after 2/3ph with no difference between genotypes. the il-6 levels gradually decline over the course of 12 hours after surgery. however, the 17c-6 mice maintained elevated il-6 at the 36-hour time point. by 168 hours (7 days) after surgery both genotypes return to baseline (figure 10(b)). a functional product of nlrc4 inflammasome activation is production and secretion of both proinflammatory cytokines il-18 and il-1 ; therefore il-1 was also measured in the plasma. we found no differences in both b6 and 17c-6 treated mice except at the 36-hour time point after surgery. at 36 hours 17c-6 has nearly an 8-fold increase over b6. by 168 hours (7 days) however, both animal groups have returned to basal plasma il-1 levels (figure 11). the ability of 17c-6 mice to resist ccl4-induced liver fibrosis could be a result of either decreased hepatic injury or increased capacity to restore hepatic function after injury. to further test restoration of hepatic induced injury by ccl4, b6 and 17c-6 mice were administered a single dose of ccl4 and allowed to recover for 24 and 48 hours. plasma alanine aminotransferase (alt) levels were measured as an indicator of hepatocyte injury. alt levels began to recover at 48 hours for both groups, with b6 averaging 965 u / l and 17c-6 averaging 431 there were no statistical differences between b6 and 17c-6 at 48 hours (figure 12). this data indicates that the degree of injury in the b6 and 17c-6 livers after acute ccl4 is similar. congenic mouse 17c-6 has shown remarkable resistance to hepatotoxin ccl4-induced fibrosis but has similar hepatocyte damage after acute exposure to this toxin. in order to replace the hepatic cells lost to this damage we measured the content of cyclin d1 mrna, a well - established marker of cellular mitosis, in b6 and 17c-6 mice given a single injection of ccl4 (figure 13). in olive oil controls we found a 9-fold increase of hepatic cyclin d1 mrna. by 24 hours the 17c-6 mice showed a 10-fold increase in hepatic cyclin d1 mrna compared to b6 mice. by 48 hours therefore, the hepatic cells in 17c-6 mice are continually in a proliferative phase, while the b6 mice match the proliferative capacity of 17c-6 at 48 hours after hepatotoxin ccl4 injection. as nafld progresses to the stage of fibrosis, the excessive deposition of extracellular matrix proteins modifies the hepatic architecture. fibrosis is the consequence of unrelenting wound - healing response after repeated injury to the liver. here we have established that 17c-6 mice have an increased regenerative liver capacity after 2/3ph (figures 611). by stimulating hepatocytes to promote cell survival and proliferation (through nlrc4 and inflammatory cytokines), the liver is capable of withstanding repeated trauma with a superior wound - healing response. this may limit the fibrotic response typical of repeated liver trauma that leads to cirrhosis and ultimately liver failure. when genetic polymorphisms are present within the coding or regulatory regions of a gene there is a potential for dysregulation of gene function leading to disease. the first was an unidentified snp (rs74459439-t) situated 331 bases upstream of the nlrc4 transcriptional start site. using transcription factor binding identification software we were able to identify this variable region of the promoter as a potential binding site for transcription factor cdx-1. chip analysis showed no interaction between cdx-1 and the b6 sequence at the snp location (figure 2). however, cdx-1 did bind to other cdx-1 binding sites within the nlrc4 promoter of the b6 mouse (figure 2, supplemental figure 1). gene expression analysis also showed increased nlrc4 expression when a promoter contained the snp from the a / j allele for nlrc4 (17c-6 mice) (figure 3). this indicates that the single base pair deletion in the a / j allele for nlrc4 promoter resulted in increased gene expression in raw 264.7 cells. overexpression of nlrc4 in human cells lines has shown increased inflammatory response to salmonella infection. also, nlrc4 overexpression leads to homooligomerization which results in mild caspase-1 activation independent of bacterial flagellin stimuli [29, 51 ]. a second snp was located within the coding region of nlrc4, in exon 5. the a / j allele contains the snp which resulted in a nonsynonymous mutation leading to an amino acid substitution (i756v), isoleucine (b6) substituted for valine (17c-6). this mutation occurs within the leucine rich repeat domain in nlrc4, a domain suggested to sequester nlrc4 in a monomeric inactivated state. others have shown that a deletion in this region of the protein results in a constitutively active nlrc4 and increased processing of il-1 [53, 54 ]. our data shows these snps display increased processing of il-1 (figure 5(a)). together, these two polymorphisms yield a mildly constitutively active nlrc4 inflammasome in macrophages of the 17c-6 congenic mouse. sterile inflammation attributable to il-1 originating from neutrophils causes an increase in il-6 secretion and acute phase proteins. this response may be blunted using inhibitors of il-1 but not caspase-1, indicating a non - caspase-1 mediated mechanism. in this case neutrophil processing of il-1 this proteinase has also been shown to participate in processing of il-18. in 2014, two independent groups of investigators published the discovery of different gain - of - function nlrc4 mutations [58, 59 ]. the patients have fever, gastrointestinal distress, and splenomegaly in the absence of any detectable infection. the disease was termed nlrc4-macrophage activation syndrome (nlrc4-mas) and shares similarity to mutations in the nlrp3 gene that result in the autoinflammatory disease, neonatal onset multisystem inflammatory disease (nomid). monocytes derived from patients with nlrc4-mas show increased il-1 secretion upon lps stimulation compared to nomid cells. nlrc4-mas monocytes and macrophages show constitutive secretion of il-18 in absence of stimuli while nomid cells show no such secretion. the gold standard to study liver regeneration is 2/3ph. in order for the liver to successfully restore the tissue lost to surgery it must orchestrate a complex endocrine signaling response from multiple cell types, initiating cellular survival and proliferation until original liver mass is restored. when challenged with 2/3ph, 17c-6 mice displayed a remarked increase in regenerative capacity (figure 6). others have shown that b6 mice restore the original liver mass within 714 days after 2/3ph [6163 ], with hepatocyte regeneration reaching a peak between 30 and 60 hours after 2/3ph. our b6 mice liver regenerative rate is in agreement with previous investigators while 17c-6 mice initiate restoration much sooner. we chose to focus on early time points of hepatic regeneration as we hypothesized that an improved ability to regenerate dead and dying hepatocytes following liver injury may be the mechanism by which the 17c-6 mice are protected from ccl4-induced fibrosis. the acute phase response is a coordinated early defense reaction in the liver responsible for protection from pathogenic infection, repair of damaged tissue, and the restoration of the proinflammatory state in response to infection and/or trauma. the acute phase response is mediated by il-1, tnf-, and il-6 cytokines and the production of acute phase proteins. designed an il-6/sil-6r fusion protein (hyper - il-6) that mimics il-6 transsignaling by directly stimulating gp130 in the absence of il-6r. after partial hepatectomy, they demonstrated that hyper - il-6, but not il-6 alone, led to early induction of hepatocyte proliferation implicating il-6 transsignaling in the regulation of liver regeneration. we hypothesize that 17c-6 mice have increased liver regenerative capacity due to elevated levels of these cytokines, leading to increased liver tissue remodeling and repair. il-18 is synthesized as an inactive precursor and must be activated through proteolytic cleavage by caspase-1. the il-18 precursor is constitutively expressed in many cell types including the resident macrophages of the liver, the kupffer cells. should nlrc4 have mild continual activation, it would repeatedly generate an active caspase-1, independent of a stimulatory trigger for the nlrc4 inflammasome activation. this active caspase-1 would in turn activate the pool of pro - il-18, thus generating proinflammatory molecules that may amplify into a full inflammatory response mediated by the generation of more pro - il-18, as well as pro - il-1 and tnf-. we have shown an increase in gene expression of all three of these cytokines after sham 2/3ph (figure 7(a)), as well as increased plasma concentrations of il-18 after sham 2/3ph (figure 10(a)). for il-18 to amplify the production of inflammatory cytokines it must bind to its membrane bound receptor and signal through the transcription factor complex nf-b. nf-b activity may be measured transcriptionally as it promotes the transcription of its inhibitor ib to serve as negative feedback. within 17c-6 mice we found a statistical increase in mrna levels of il-1, il-18, tnf-, and ib relative to b6 mice (figure 7(a)). interestingly, il-1 protein levels in plasma do not increase until hour 36 after surgery in 17c-6 mice. we postulate that this 36-hour lag period may be attributed to the time needed to amplify the localized inflammatory response and secrete enough il-1 that is detectable in the plasma. the replacement of hepatic cells lost to infection, trauma, and inflammation is primarily mediated through il-1 family cytokines, tnf-, and il-6 (all transcriptional targets of nf-b [7073 ]) through their induction of the inflammatory acute phase response. a key regulator of hepatocyte regeneration, il-6, is produced as a consequence of the inflammatory signaling cascade initiated by the nlrc4 inflammasome. downstream targets of il-6 signaling (c - fos, c - myc, crp, and cyclin d1) participate in hepatocyte survival and proliferation. we demonstrated that 17c-6 mice have increased il-6 transcription levels in the sham surgery liver as well as systemically increased plasma il-6. therefore il-6 carries out signaling from the macrophage to the hepatocyte by interaction with the membrane receptor complex il-6r subsequent signaling through stat3 results in transcriptional activation of acute phase response proteins such as crp, generation of cell - cycle regulators including cyclin d1, as well as transcription factors in control of cellular differentiation and cycle progression, c - myc and c - fos [7476 ]. 17c-6 mice have increased mrna concentrations for stat3, cyclin d1, c - fos, and c - myc in liver tissue obtained in the sham surgery and increased phosphorylation of stat3 after ph (figure 7(b)). this indicates a strong proliferative signal within the hepatocytes, even before the liver experiences trauma. the cells are primed for a quick reaction to liver damage with an increased ability to resolve this damage, diminishing the overall effects of hepatic trauma. this potentially contributes to the decreased susceptibility to fibrosis development seen in congenic mouse 17c-6. previously we have published that 17c-6 mice were resistant to ccl4-induced liver fibrosis. for 17c-6 to have a marked decrease in accumulation of extracellular matrix proteins (fibrosis) the mice must have either reduced hepatotoxin - induced injury or an amplified ability to regenerate or increased ecm turnover as well as other possibilities. to fully understand this decrease in fibrosis susceptibility, b6 and 17c-6 mice were acutely exposed to ccl4. 17c-6 mice given a single dose model of carbon tetrachloride displayed hepatic injury comparable to b6 mice. however we found increased hepatic mitosis in 17c-6 mice (figure 13). in figure 14 the a / j allele for nlrc4 has two snps that result in chronic low level activation of nlrc4 in the kupffer cells. in the basal state the 17c-6 mice secrete increased il-18 due to the chronic activation of nlrc4 (figure 10(a)). il-18 binds to its receptor in the kupffer cell and initiates signaling to activate nf-b signaling which in turn increases expression of il-6, tnf-, il-18, and il-1. the elevated il-6 binds to il-6 receptor / gp130 in the liver and activates jak / stat signaling. the activated stat3 induces cyclin d1, crp, c - myc, and c - fos transcription, which together, increase the rate of hepatic cell proliferation, differentiation, and survival. by characterizing the nlrc4 inflammasome and its association to liver regeneration here we have identified that an nlrc4 inflammasome - driven production of inflammatory cytokine signaling leads to a coordinated hepatoprotective response to ccl4-induced and hepatectomy - induced liver damage. the ability of inflammatory cytokines tnf-, il-1, and il-18 to stimulate hepatocyte proliferation, mediated through il-6, permits for a flexible restoration network to repair liver tissue after trauma. further investigation is necessary to research the signaling pathways responsible for hepatic regeneration, specifically their intercellular signaling cascades and subsequent cellular responses. this study validates that constitutive activation of the inflammasome to produce mature interleukins il-18 and il-1 within the liver leads to the increased production and secretion of il-6, a key regulator of liver regeneration. this mild, continuous inflammatory response was shown to diminish the development of hepatotoxin - induced fibrosis as well as facilitate the enhanced regeneration of liver mass after hepatectomy. the ability to promote healing within a damaged liver has direct clinical implications for the prevention and/or treatment of diseases of the liver. | tthe molecular mechanisms responsible for the development of hepatic fibrosis are not fully understood. the nlrc4 inflammasome detects cytosolic presence of bacterial components, activating inflammatory cytokines to facilitate clearance of pathogens and infected cells. we hypothesized that low - grade constitutive activation of the nlrc4 inflammasome may lead to induced hepatocyte proliferation and prevent the development of hepatic fibrosis. the gene of nlrc4 contains two single nucleotide polymorphisms (snps), one located within the nlrc4 promoter and one contained within exon 5. these snps regulate nlrc4 gene transcription and activation as measured through gene reporter assays and il-1 secretion. the 17c-6 mice have increased il-1 in plasma after chronic carbon tetrachloride (ccl4) administration compared to b6 mice. after two - thirds partial hepatectomy (2/3ph) 17c-6 mice have earlier restoration of liver mass with greater cyclin d1 protein and brdu incorporation compared to b6 mice at several time points. these data reveal mild constitutive activation of the nlrc4 inflammasome as the results of two snps, which leads to the stimulation of hepatocyte proliferation. the increased liver regeneration induces rapid liver mass recovery after hepatectomy and may prevent the development of hepatotoxin - induced liver fibrosis. |
the present study investigates the interaction of aqueous leaf extract of psidium guajava with muscarinic, serotonergic and adrenergic receptor system using isolated rat ileum, gastric fundus and trachea, respectively. the concentration - dependent contractile response of aqueous leaf extract of psidium guajava was parallel and rightward of standard agonists, ach and 5-ht indicating agonistic activity on muscarinic and serotonergic receptor systems. the inhibition of aqueous leaf extract of psidium guajava mediated contractions in presence of atropine (10 - 7 m) and ketanserin (10 - 6 m) confirmed the activity. relaxant effect of pg (0.2 mg / ml) on carbachol induced pre - contracted rat tracheal chain indicated its agonistic action on adrenergic receptor system. inhibition (p<0.05) of the action in the presence of propranolol (1 ng / ml) confirmed the activity. it may be concluded that pg possesses agonistic action on muscarinic, serotonergic and adrenergic receptor systems. |
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dermatomyositis (dm) is an idiopathic inflammatory myopathy with progressive, symmetrical weakness of the proximal muscles and characteristic cutaneous manifestations such as poikiloderma. the etiology and pathogenesis of dm remain unknown ; however, in recent years, some researchers have shown that the cause of dm may be related to an autoimmune response induced by a viral infection or a paraneoplastic syndrome. in particular, dm has been strongly associated with breast, ovarian, lung, pancreatic, gastric, and colorectal cancer, as well as non - hodgkin 's lymphoma. however, dm associated with hepatocellular carcinoma (hcc) has rarely been reported [2 - 7 ]. here, we report a case of dm associated with hepatitis b virus (hbv)-related liver cirrhosis and hcc with a review of the current literature. a 55-year - old male visited our hospital complaining of progressive weakness in his bilateral arms, hips, and thighs. he had difficulty combing his hair and was experiencing dysphagia and erythematous changes in the skin (his face and a ' v ' on his upper chest and neck that had developed 3 weeks earlier) (fig. 1). a physical examination revealed a heliotrope rash with bilateral periorbital edema and poikiloderma on the upper chest and neck. serum laboratory tests revealed the following : white blood cells 9,720/mm, hemoglobin 13.6 g / dl, platelets 98,000/mm, creatine kinase 16,440 iu / l (normal range, 0 to 190), lactate dehydrogenase 994 iu / l, aspartate aminotransferase 717 iu / l, alanine transaminase 139 iu / l, total bilirubin 1.4 mg / dl, albumin 2.2 g / dl, alkaline phosphatase 119 iu / l, and prothrombin time (activity) 55%. hbv surface antigen was detected, and the hbv dna level was 7,380 iu / ml. no antibodies against hepatitis c virus were detected. igm antibodies against epstein - barr virus, cytomegalovirus, and herpes simplex virus were not detected. tests for other autoantibodies, including anti - dsdna, anti - ribonucleoprotein, anti - sm, anti - jo-1, and anti - scl70, were all negative. electromyography showed a small number of polyphasic waves with a normal interference pattern in all muscles tested. a muscle biopsy of the right upper arm showed a sparse lymphocytic infiltrate around the blood vessels and negative immunofluorescence pathologic findings, compatible with dm (fig. the patient 's levels of carcinoembryonic antigen, carbohydrate antigen 19 - 9, and prostate - specific antigen were normal, but he had elevated levels of -fetoprotein (afp ; 448.8 ng / ml) and proteins induced by vitamin k absence - ii (> 500 mau / ml). liver dynamic computed tomography (ct) revealed a 6.5-cm sized arterial enhancing mass in hepatic segment 7/6 (fig. we diagnosed the patient with hbv - related hcc based on his status as an hbv carrier, elevated afp level (> 200 ng / ml), and typical radiographic findings (e.g., arterial enhancing features on liver dynamic ct). optimal management procedures for hcc such as transcatheter arterial chemoembolization could not be performed because of the patient 's poor general condition. after steroid therapy, the patient 's skin rash, muscle strength, and dysphagia showed slight improvement, and his elevated muscle enzyme levels returned to the near - normal range. however, his underlying hcc progressed rapidly : the size of the cancer grew up to 11 cm with portal vein thrombosis and multiple lymph node metastasis. an association between malignancy and dm has been widely reported in the literature with incidences ranging from 3% to 35%. possible mechanisms include paraneoplastic syndrome, a compromised immune system, common carcinogenic environmental factors, and cross - activity of immune reactions against the tumor, all of which transform into an autoimmune syndrome as a consequence of cross - reactivity with skin and muscle antigens. levine suggested a model of paraneoplasia focusing on common autoantigen expression and immune targeting between cancer tissues and muscle tissue in myositis. one of these so - called myositis - specific autoantibodies recognizes mi-2 antigen, a component of the nucleosome remodeling deacetylase complex. recent data examining the immune recognition of mi-2 antigen suggest that patients with cancer - associated myositis develop humoral immune responses against different regions of the molecule, as compared to the responses of those with myositis alone. proposed that anti - mi-2 antibodies may be cross - reactive with hcc. in our case, there was another possibility - that the patient 's dm was caused by other infectious agents. therefore, we tested for various infectious agents, including echovirus, adenovirus, coxsackie virus, influenza, human immunodeficiency virus, and human t cell leukemia / lymphoma virus, which are known to cause myositis, and all tests were negative. antibodies that attack a virus or virus - enzyme complex could cross - react with a homologous area of host proteins and result in autoantibody production (i.e., a cross - reactive phenomenon). a test for anti - jo-1 antibodies was negative, but anti - mi-2 antibodies were not examined in this case. six patients with dm associated with hcc have been described in the literature including the present case (table 1) [2 - 7 ]. these results suggest that dm in patients with hcc is a risk factor for advanced hcc. dm has been noted to improve after the treatment of cancer, with recurrences of muscle weakness taking place after relapse of the malignancy, further suggesting a paraneoplastic origin. unfortunately, we could not manage the hcc in our patient because of his poor general condition and hepatic function. there was a previous report of improvement in dm without corticosteroids after the resection of hcc. in our case dm may be caused by hcc, especially advanced hcc, and/or a chronic hbv infection. therefore, hcc might be considered a cause of dm if no other etiology can be determined. | dermatomyositis is an idiopathic inflammatory myopathy with typical cutaneous manifestations. it has been proposed that dermatomyositis may be caused by autoimmune responses to viral infections. previous studies have shown an association between dermatomyositis and malignant tumors such as ovarian cancer, lung cancer, and colorectal cancer. however, a chronic hepatitis b virus (hbv) infection associated with dermatomyositis and hepatocellular carcinoma (hcc) has been very rarely reported. here, we report a rare case of dermatomyositis coinciding with hbv - associated hcc. a 55-year - old male was confirmed to have hcc and dermatomyositis based on proximal muscle weakness, typical skin manifestations, elevated muscle enzyme levels, and muscle biopsy findings. this case suggests that hcc and/or a chronic hbv infection may be factors in the pathogenesis of dermatomyositis through a paraneoplastic mechanism. |
cardiac surgery is connected to profound inflammatory response, characterized by increased level of both proinflammatory and anti - inflammatory mediators. cardiopulmonary bypass (cpb) is considered to be a potential trigger of cytokine release, therefore, the impact of cardiac surgery on morbidity and mortality is often correlated with the use of cpb [1, 2 ]. surgery conducted with the use of cpb is accompanied by ischemia - reperfusion injury, and above that, cpb also represents the environment where blood cells become activated by contact with artificial surfaces, direct air - contact, and also nonturbulent flow. the effort to remove the harmful effect of conventional cpb has led to a development of new surgical devices and techniques. mini - cpb, that has recently been introduced and successfully used, is designed to reduce blood cell activation : it provides decreased area of extracorporeal circuit, reduces priming, and also lessens air - blood interface. another favorable feature of mini - cpb is a biocompatible coating that induces higher tolerance by blood cells. the impact of mini - cpb on suppressing inflammatory response is already described and published [3, 4 ]. as the blood reaches all the body compartments, inflammatory response to surgical injury exceeds local reaction and becomes systemic. although the increased production of cytokines is essential for protection against infection and also healing the wounds, unregulated inflammatory response is likely to have a harmful effect. regulating mechanisms consist of anti - inflammatory molecules such as cytokines, cell surface enzymes, receptor antagonists, and soluble receptors. the net impact of the produced cytokines, whether with proinflammatory or anti - inflammatory properties, is determined by the ability of the immune system to balance the inflammatory response properly. il-10 is a regulatory and immunosuppressive cytokine that is produced by a variety of cells. upon binding to its receptor (il-10r), il-10rb subunit transmits a signal into the cell which activates stat3- and stat3-resposive genes. as a result, the production of proinflammatory cytokines, such as il-1 and tnf-, is inhibited. the changes in production of il-10, il-1, and tnf- have already been extensively evaluated in patients undergoing cabg surgery with conventional cpb. however, the information about kinetics of il-10 in mini - cpb patients is sparse. we compared the changes of il-10 serum level, the expression of il-10 membrane receptor, and the percentage of neutrophils in conventional and mini - cpb patients, as well as between these groups of patients. forty - four patients, undergoing elective coronary artery bypass grafting (cabg) surgery on an arrested heart using cpb, were enrolled into our study. all patients were well informed about the purpose of the study and they confirmed their unconstrained participation by a written consent. the study project was approved by the ethics committee of the university hospital in hradec kralove, czech republic. patients, included in the study group in the period from december 2006 to december 2007, were randomly assigned to surgery either with the use of conventional cpb (n = 22) or mini - cpb (n = 22). exclusion criteria consisted of acute inflammation, urgent operation, reoperation, combined operations, operative risk more than 5% (according to logistic euroscore), preoperative level of serum creatinine above 130 mol / l, hepatic disease, and malignancies. cpb was established using a two - stage venous drainage and ascending aortic return. a roller pump (stckert instrumente gmbh, mnchen, germany), a membrane oxygenator (dideco srl, mirandola, italy) in a closed modification with a collapsible reservoir, a cardiotomy suction device, and a 40 m arterial line filter (dideco srl, mirandola, italy) were integrated into the extracorporeal circuit. the priming solution consisted of 500 ml of ringer 's lactate, 500 ml of rheodextran (rheomacrodex), 5,000 iu heparin, 80 ml of natrium bicarbonate (nahco3 8,4%), 20 ml of 10% magnesium sulphate, and mannitol (at 1 g / kg body weight). heparin was administered intravenously at 300 iu / kg body weight to maintain an activated clotting time (act) above 480 s during bypass procedure. pump flow rates averaged 2.4 l / min / m of body surface area with pressure maintained at 5060 mmhg. cardioplegic arrest was induced with a cold blood cardioplegic solution, which consisted of blood mixed with st. thomas solution (ardeapharma, sevetin, czech republic) in 4 : 1 ratio. it was administered antegradely into the aortic root with doses added every 20 min or as needed. all patients received an internal artery mammary graft to the left anterior descending coronary artery. the central aortovenous anastomoses were performed during the reperfusion phase of cardiopulmonary bypass with the heart beating. after the termination of cpb, heparin anticoagulation was antagonized using protamine sulphate at a dose of 1 : 1. mini - cpb was established using a small 22f two - stage venous drainage and ascending aortic return. minisystem synergy (sorin group srl, mirandola, italy) consisted of a centrifugal pump, membrane oxygenator, 40 m arterial line filter, and a venous bubble trap. the whole system, consisting of a closed loop with the surface treated with ph.i.s.i.o phosphorylcholine coating (sorin group srl, mirandola, italy) and very short tubing, was placed close to the operating field. the priming solution, heparinization, pump flow, temperature, and surgery technique were identical with the conventional cpb procedure described above. cardioplegic arrest, induced according to the calafiore warm blood - cardioplegia protocol, was administered antegradely into the aortic root. at the beginning of cpb, crystalloid priming solution was flushed retrogradely together with the blood coming from the arterial line to minimize the hemodilution of the patient. mean arterial pressure was kept above 50 mmhg, with norepinephrine administered as required. blood samples were withdrawn from subclavian vein before and during surgery and from antebrachial vein in postsurgery period. the samples were collected into anticoagulant - untreated vacutainer tubes as well as heparinized vacutainer tubes (becton dickinson, uk) at the following time points : before surgery (introduction of anesthesia), at the beginning of cpb, at termination of cpb, at the end of surgery, and on the 1st, 3rd, and 7th postoperative day. serum was separated from the blood cells by centrifugation at 1000 g for 15 min. il-10 concentration was determined by enzyme - linked immunosorbent assay (elisa), with a sensitivity of 1 pg / ml. elisa kit was purchased from bender medsystems (austria). il-10 concentrations were measured using spectrophotometer (labsystems multiskan rc, usa) with genesis software. heparinized blood samples were processed immediately after collection. 50 l of blood was incubated with titrated monoclonal antibodies anti - cd3 fitc / il-10r pe / cd14 percp / cd16 apc. antibodies, except for anti - il-10r, were purchased from bd biosciences (usa), while anti - il-10r was purchased from biolegend, usa. following the 20 min incubation, red blood cells were lysed by hypotonic lysis and then the samples were washed. the data acquisition was performed with facs calibur flow cytometer using cellquest software (bd biosciences, usa), and the analysis was done using flowjo software (tree star, usa). values of il-10, il-10r, and percentage of neutrophils during and after surgery were compared to preoperative values. changes within a group were evaluated using anova for repeated measures and dunnett 's test or friedman anova and wilcoxon paired test. to determine the differences between both groups of patients, values of il-10, il-10r, and percentage of neutrophils were compared at matching time points using two - way anova for repeated measures and fisher 's lsd test or mann - whitney u test. demographic and clinical data were analyzed by fisher exact test, mann - whitney u test, and student 's t - test. changes in il-10 serum level were similar in both groups of patients, with respect to the preoperative serum level of 1 pg / ml in conventional cpb group and 1.45 pg / ml in mini - cpb group. in the conventional cpb group, serum level of il-10 significantly increased at the termination of cpb (52.7 pg / ml, p < 0.001), at the end of surgery (50.5 pg / ml, p < 0.001), and on the 1st postoperative day (7.3 pg / ml, p similarly, the mini - cpb group experienced an increase in serum level at the termination of cpb (from 1.5 pg / ml to 10.6 pg / ml, p < 0.01), at the end of surgery (21.2 pg / ml, p < 0.001), and on the 1st postoperative day (5.8 pg / ml, p < 0.05). postsurgery monitoring revealed that the level of il-10 was gradually decreasing in both groups and did not significantly differ from preoperative levels (figure 1). there was significant serum - level difference (p < 0.01) between both groups at the termination of cpb : the conventional cpb group topped the mini - cpb group by reaching the value of 52.7 pg / ml. at this time point, il-10 in mini - cpb group was enhanced, but only reached 10.6 pg / ml. in mini - cpb group, the level of il-10 was highest at the end of surgery (21.2 pg / ml). in postsurgery period, the level of il-10 decreased and did not differ between both groups (figure 1). percentage of neutrophils was significantly increased (p < 0.001) in both groups of patients at the end of cpb, at the end of surgery, and also on the 1st and 3rd postoperative day. the baseline was 55% of neutrophils in conventional cpb group and 53% in mini - cpb group. the highest percentage of neutrophils was measured on the 1st postoperative day in both groups of patients (80% in conventional cpb group and 81% in mini - cpb group). there was statistically significant difference in percentage of neutrophils between both groups at the end of surgery (p < 0.05) when conventional cpb group reached 79% and mini - cpb group reached 75% of neutrophils (figure 2). the percentage of neutrophils correlated with the maximum level of il-10 at the termination of cpb (rs = 0.73, p < 0.001), at the end of surgery in conventional cpb group (rs = 0.54, p < 0.01), and at the end of surgery in mini - cpb group (rs = 0.54, p < 0.01) (figures 3(a)3(c)). since il-10r is expressed on t lymphocytes, neutrophils, and monocytes, the expression was statistically evaluated in all of these populations. in lymphocytes and neutrophils, no differences were found at any time point when comparing mfi values within a group of patients or between both groups of patients. the expression of il-10r on monocytes significantly decreased at the end of surgery in both groups : the preoperative expression of il-10r dropped from mfi of 10.2 to 7.6 (p < 0.01) in conventional cpb group, as well as in mini - cpb, where the preoperative level dropped from mfi of 9.8 to 7.6 (p < 0.05). on the 3rd postoperative day, the expression of il-10r on monocytes was significantly enhanced (figure 4), reaching mfi of 10.6 in conventional cpb (p < 0.05) and 11.3 in mini - cpb (p < 0.05). after that, the il-10r expression decreased again and there was no significant difference between the preoperative value and the value on the 7th day after surgery in both groups (figure 5). there was no correlation between serum level of il-10 and expression of il-10r on monocytes. no difference in il-10r expression on monocytes was observed when comparing both groups of patients (figure 5). we observed single- as well as multiple - organ dysfunction or failure in both groups of patients. il-10 is an important cytokine, maintaining the proinflammatory response balanced. although il-10 can keep cells unresponsive, receptors on the cells affected by il-10 are still capable of binding and internalizing proinflammatory cytokines. as a result, the cytokines are removed from blood circulation without subsequent activation of the cells [11, 12 ]. this kind of decoy activity is thought to be of favorable contribution of il-10 in ischemic heart after myocardial reperfusion, and it is likely to prevent against heart failure. however, high level of il-10 can also have an adverse effect by increasing the likelihood of sepsis. nonetheless, a significant association between sepsis and higher level of il-10 determined by1082 base pair single polymorphism in promoter region of il-10 gene is equivocal [14, 15 ]. it has been reported that cpb induces the release of il-10, which can be further enhanced by the use of corticosteroids in patients undergoing cabg. in our study, corticosteroids were not applied, and yet we observed that cpb induced a profound release of il-10. we found significant difference in il-10 production between the two different types of cpb used. mini - cpb, which is considered to be a less harmful approach of cardiac surgery, elicited a lower release of il-10. in spite of this fact although the level of il-10 was lower in mini - cpb group, the number of patients suffering from organ dysfunction or failure was nonsignificantly higher in mini - cpb group than in conventional cpb group (p < 0.22) (table 2). while we would be able to predict organ dysfunction or failure according to il-10 level in conventional cpb group, the similar relation was unclear in mini - cpb group. we also looked at the il-10 value in patients with sepsis and patients without sepsis. microbiologically confirmed, there were only two septic patients, one in each group, both reaching values of il-10 in serum that fell within quartiles of il-10 calculated for each sampling time point in any given group. therefore, in our study groups, even though the il-10 level was very high in some patients, il-10 could not be used as a predictive marker related to sepsis. increased level of il-10 along with the medical treatment (ampicillin - sulbactam) of our patients might have prevented the onset of sepsis. such a beneficial role of il-10 is in concordance with experimental studies [17, 18 ]. in previous works, il-10 level was also found to be significantly increased in patients who suffered from postoperative renal dysfunction, which was characterized by creatinine level raised above 176 mol / l. the mini - cpb group had two patients that were suffering from acute renal dysfunction, while there was only one patient in conventional cpb group. the patient from conventional cpb group reached 522.5 pg / ml at the termination of cpb.. however, the other two patients did not exceed quartiles of il-10 calculated for each sampling time point. according to this result, there is no simple relation between enhanced level of il-10 and increased probability of acute renal dysfunction. we compared the percentage of neutrophils to il-10 level when il-10 in serum was at its highest level. we observed significant correlation between il-10 and percentage of neutrophils at the termination of cpb in conventional cpb group (rs = 0.73, p < 0.001) and at the end of surgery in mini - cpb group (rs = 0.54, p < 0.01). we also found lower but still significant correlation (rs = 0.54, p < 0.01) between il-10 and percentage of neutrophils at the second highest level of il-10 (at the end of surgery) in conventional cpb group. our data suggests that neutrophils were the main producers of il-10 in most of our cardiac surgical patients. however, the lower correlation coefficient at the end of surgery indicates other cells might also have participated in il-10 production. the observation is in agreement with recently published findings that show that neutrophils are an important source of il-10 and that mini - cpb attenuates neutrophil activation and cytokine release after coronary bypass surgery. it seems that higher level of il-10 in conventional cpb group is exclusively linked to the surgery technique and devices used. since we found no significant difference in expression of il-10r between both groups of patients, we can hypothesize that the expression of il-10r on hematopoietic cells exceeds the maximum level of its ligand, il-10. it has been discovered that other cells, such as fibroblasts and epithelial cells [23, 24 ], also express il-10ra after induction ; thus il-10 may have a much broader effect on tissues and organs, an effect which can not be explained by the possible correlation of serum il-10 and the expression of il-10r on monocytes. il-10 level and percentage of neutrophils are significantly affected by the type of cardiac surgery employed. although il-10 level may have statistical relation to sepsis or renal dysfunction, the generally accepted critical level that would enable to unambiguously distinguish between patients with worse or good prognosis does not exist. however, in certain conditions, like using conventional cpb, il-10 may represent a supporting tool, that, along with other parameters, would help rank the patients in likelihood of organ dysfunction or failure. the observed correlation between increased level of il-10 and higher percentage of neutrophils in both groups of patients suggests functional relationship between both parameters. | interleukin-10 (il-10) is considered to be a cytokine with potent anti - inflammatory properties, which have been previously linked to increased incidence of sepsis. the level of il-10 is elevated by cardiac surgery when cardiopulmonary bypass (cpb) and methylprednisolone are used. in our study, we compare the level of il-10, il-10 receptor (il-10r), and percentage of neutrophils between two groups of cardiac surgical patients undergoing coronary artery bypass grafting, both of which were not given methylprednisolone. the first group was operated with conventional cpb, while the second group was operated with minimally invasive cpb (mini - cpb). we detected enhanced level of il-10 during surgery and at the end of surgery in both groups of patients. while no correlation between il-10 and il10r was found, il-10 was positively correlated with increased percentage of neutrophils at the time points when the level of il-10 peaked. |
the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. | experimental investigations of the neural substrate of consciousness typically take one of two paths, studying (1) contents or (2) levels of consciousness. it seems obvious to most that these two paths are interrelated, yet much less obvious how. this paper gives one suggestion to grasp the interrelation, arguing that conscious levels are determined by conscious contents in a very specific way. it follows from the argument that conscious contents are so - called natural kinds, whereas conscious levels are not. |
smaller displacement of the global center of pressure with less body weight bearing on the feet results in less displacement of center of mass (com) during trunk flexion in persons with hemiplegia1. postural control is the capability to control the body weight by keeping com within of the base of support (bos)2. patients with hemiplegia after a stroke have difficulty managing their com with their bos. by having the patients using a posterior pelvic tilt during trunk movements, those factors restrain the functional performance, such as a decreased range of motion and stiffness1. the contributing factors which should be achieved are the postural tone regulation, particularly in the extensor antigravity musculature and correct foot placement3. corticospinal system damage can generate long - term failure of the intrinsic musculature activation in the foot. so, it is necessary to gain ability to extend the toes for the selective dorsi - flexion. the soleus is another factor contributing to poor dorsi - flexon of the foot unless it is proper lengthened and strengthened as an antagonist4. so, facilitated ankle dorsi - flexion of the affected foot influences trunk range of motion when not only flexing forward, in particular, but also with the backward flexion of the trunk in a sitting position. therefore, this study intends to reveal the relationship among ankle dorsi - flexion, spinal range of motion and gait ability. in this study, there was a gait group (gg) of 6 subjects including 5 males and 1 female with the mean age of 61 years, and a non - gait group (ngg) of 6 subjects including 4 males and 2 females with the mean age of 63 years old, all participants with hemiplegia after a stroke. the subjects in the gg were able to walk by themselves but the subjects in the ngg were not. five patients had left hemiplegia and 1 patient had right hemiplegia with a mean of 12 months of time after a stroke in the gg : four patients had left hemiplegia and 2 patients had right hemiplegia with 27 months of it in the ngg. there were no significant differences in the homogeneity between 2 groups in general characteristics. inclusion criteria were as follows : (1) unilateral stroke with hemiparesis ; (2) medical stability ; and (3) capability to comprehend the test procedures. both groups were gathered from d hospital in jeonjoo and this study took place from november 17 to december 19, 2014. the test was conducted before and immediately after the intervention and 30 minutes later. the subjects were provided informed consent documents to take part in the study prior to its commencement and this study followed the principles of the declaration of helsinki. the spinal mouse (idiag, volkerswill, a physical therapist who had 11 years of clinical experience treated the subjects with the intervention for 30 minutes just once per each subject. all subjects were informed about the procedure to make sure of the subjects safety before applying the intervention. at first, the researchers made sure the patient would be in an optimal sitting position on a plinth. after stretching the intrinsic muscles of the affected foot, the tibialis anterior, extensor hallucis longus, extensor digitorum longus were facilitated using a combination of distraction, compression and movement of toes to get the toe extension involved. the next step was making the affected knee flex and extend repeatedly with the ankle eversion plus dorsiflexion to activate the distal part of the rectus femoris. all data was analyzed using spss version 18 (statistical package for the social science). variations in spinal parameters obtained by spinal mouse within each group were tested with the friedman test. a =0.05 level of significance the sacral hip angle in the gg increased from 32.4 11.7 in pre - test to 40.3 10.9 in post - test to 41.4 9.0 in follow - up when upright flexion was used inclination increased from 71.9 7.9 in pre - test to 78.3 6.3 in post - test to 80.5 11.3 in follow - up in the gg when upright flexion was used as well. when flexion extension was used, sacral hip angle increased from 36.3 9.0 to 38.4 12.7 to 50.9 13.5 and inclination increased from 80.0 7.9 to 87.1 8.3 to 88.3 14.5 in pre, post and follow - up test in the gg. however, lumbar spine angle decreased significantly from 42.6 8.8 to 36.1 12.0 to 35.3 14.1 in pre, post and follow - up test in the gg when flexion extension was used (table 1table 1.variation of spinal range of motion in the sagittal plane in sitting (n=12)()gg ngg prepostfuprepostfuu - fsh 32.411.740.310.941.49.033.623.734.722.131.518.0ts 31.913.831.712.121.518.718.622.019.127.415.616.6ls 38.014.637.79.938.611.236.813.932.513.235.710.7i 71.97.978.36.380.511.370.115.868.018.567.219.4u - esh 4.06.98.26.39.58.51.54.71.54.51.23.7ts 0.38.95.411.66.78.71.839.15.07.011.916.8ls 4.69.51.57.13.48.14.08.11.24.93.33.9i 8.02.68.93.27.85.02.22.93.01.12.02.5f - esh 36.39.038.412.750.913.532.325.336.622.630.320.0ts 31.614.237.08.628.019.920.520.524.127.427.611.4ls 42.68.836.112.035.314.1 40.916.333.711.139.011.0i 80.07.987.18.388.314.572.114.771.118.169.120.3p<0.05 ; meansd ; sd : standard deviation ; sh : sacral hip ; ts : thoracic spine ; ls : lumbar spine ; i : inclination ; uf : uprightflexion ; ue : uprightextension ; fe : flexionextension ; gg : gait group (n=6) ; ngg : nongait group (n=6) ; fu : follow up). p<0.05 ; meansd ; sd : standard deviation ; sh : sacral hip ; ts : thoracic spine ; ls : lumbar spine ; i : inclination ; uf : uprightflexion ; ue : uprightextension ; fe : flexionextension ; gg : gait group (n=6) ; ngg : nongait group (n=6) ; fu : follow up in a previous study, spinal range of motion (rom) tended to increase down the vertebral column in flexion extension (fe). and l5s1 fe rom were significantly greater than for every other level (p<0.003)6. as in the previous study, the lower part of vertebral body is much more mobile than others. sacral hip angles in both upright flexion and flexion extension increased gradually in the gg in this study. this is because the hip joint is a main joint not only for bearing body - weight, but also for giving the most mobility when bending the trunk forward and backward while sitting. even though the lumbar spine angle decreased by about 6.5 and 0.84 degrees little by little, the sacral hip angle compensated for the amount of the decrease by increasing 2.09 and 12.49 degrees steadily in the gg. it had the same result in upright extension as that in upright flexion in the gg. this has an important implication, in that lumbar spine angle and sacral hip angle have a close relationship to one another when trying to bend the trunk forward and backward. if the lumbar spine angle is smaller than before, then the sacral hip angle becomes larger than what it was for compensation. in addition, inclination in flexion extension increased gradually by 7.17 and 1.16 degrees in each test in the gg. first, the inclination is closely interrelated with the sacral hip angle positively which means that, as the results have shown sacral hip angle influences the extent of trunk mobility in sitting position. second, increased total range of inclination means that the subjects have a decreased possibility of falling down when compared to subjects with a lesser range of inclination. in a previous study, the falls groups have a considerably decreased mobility of spinal inclination as compared to those in the non - falls group7. in clinical insight, inclination is much more important than other factors when it comes to postural stability. to create a more stable posture, we need wider stability limit which means that we have to have an ability to transfer the center of mass as far as possible without a change of base of support. therefore, this study suggests that subjects who have bigger sacral hip range can bend their trunk forward and backward farther. as a result, more pay attention should be paid to the mobility of sacral hip in order to get better postural control in the sitting position. | [purpose ] the purpose of this study was to determine alterations of spinal range of motion while sitting, in hemiplegic patients with or without gait available. [subjects ] there was a gait group (gg) of 6 subjects, and a non - gait group (ngg) of 6 subjects, both with hemiplegia after a stroke. [methods ] the subjects in both groups were given an intervention focusing on ankle dorsi - flexion of the affected foot only once for 30 minutes. the spinal mouse was used to gain data of the spinal range of motion before and after the intervention and 30 minutes later for follow - up test. [results ] only in the gait group, lumbar spinal range of motion showed a significant difference when using flexion extension. sacral hip and inclination were both increased gradually when upright flexion and flexion extension were used. [conclusion ] facilitating foot for ankle dorsi - flexion is effective on spinal range of motion especially sacrohip, lumbar spine and inclination only for the subjects in the gait group. the results suggested that ankle dorsi - flexion exercise influences spinal range of motion in a sitting position. |
we used the 2007 marketscan commercial claims and encounters (cce) database (marketscan database ; thompson medstat, ann arbor, mi). this database, which has been used extensively in studies of health care costs, including studies of diabetes costs (12,1416), contains fully adjudicated and paid claims for several million people with employer - sponsored health plans, including employees, spouses, and dependents (17). the cce database includes patient - level data on inpatient, outpatient, and drug claims that can be linked using encrypted enrollee information. types of health plans from which enrollees obtain services can be divided into fee - for - service (ffs) plans and fully or partially capitated plans. ffs plans include preferred provider organization (ppo) plans, exclusive provider organization plans, point - of - service (pos) plans, consumer - directed health plans, and indemnity plans. fully or partially capitated plans include hmo plans and capitated pos plans (18). cost estimation for those enrolled in ffs health plans for those enrolled in capitated plans, payment records often reflect encounter status and not the actual payment (18). youth who had health care service and prescription drug coverage through an ffs plan between 1 january and 30 december 2007. first, following criteria used in a previous study (19), enrollees were identified as having diabetes if during calendar year 2007 they had at least one claim for diabetes - related drugs and supplies and at least two outpatient encounters 30 days apart in which diabetes was a primary or secondary diagnosis or at least one inpatient admission with diabetes as a primary or secondary diagnosis. the requirement of at least two outpatient encounters excludes those who were misdiagnosed at the first encounter but later determined not to have diabetes (16). we used the icd-9-cm codes 250.00250.93, 357.2, 362.0362.02, and 366.41 (18) as diabetes indicators in the outpatient and inpatient claims. in the second step, youth with dm were identified to have itdm if they had at least one drug claim for insulin as indicated by therapeutic class code 172. last, youth with itdm were identified to have dka if they had any claims coded with icd-9-cm code 250.1 (2) and with severe hypoglycemia if they had any claims coded with icd-9-cm codes 251.0, 251.1, 251.2, or 250.8 (12). to determine the extent to which the number of episodes of dka or severe hypoglycemia was associated with excess expenditures, we dichotomized youth with these complications into those having one episode and those having more than one episode. we considered an individual to have had one episode of dka if they received one service with a dka code during the study period and to have had more than one episode if they had received at least two such services at least 30 days apart. we excluded 39 with missing census region or urbanity of residence (urban versus rural). we also excluded 129 people with uncommon chronic conditions (congenital heart failure, hemiplegia, lymphoma, down 's syndrome, autism, leukemia, liver diseases, and congenital heart defects). we could not consider these conditions because the number of youth experiencing each condition was very small (e.g., 10 among dka or severe hypoglycemia youth). thus, our final analytic database had 7,556 youth. because of its relative high prevalence and possible interaction with diabetes expenditures we used stata version 10.1 (stata, college station, tx) for all analyses. we estimated four expenditure models (outpatient, inpatient, drug, and total). the presence or absence of dka or severe hypoglycemia covariates included age, sex, census region (midwest, south, west, and northeast), residence urbanity, health benefit plan type (ppo versus non - ppo), and presence of asthma. we compared the unadjusted means of the characteristics of youth by their dka or severe hypoglycemia status with student t tests. we used a generalized linear model with log link and gamma distribution to model total, outpatient, and drug expenditures because all youth had positive expenditures and expenditures were highly skewed and right tailed (20). we used a two - part model to model inpatient expenditures to account for those with no inpatient expenditures (21,22). in the first part of the two - part model, we used logistic regression to estimate the probability that someone would have had any inpatient expenditures, and, in the second part, we used a generalized linear model with log link and gamma distribution to estimate the inpatient expenditures for those who had such expenditures. we calculated model - based predicted marginal medical expenditures by dka status. in this approach, predictions were made for all observations assuming no dka status for all observations and then again assuming dka. the predicted excess expenditure associated with dka was calculated as the difference between the predicted expenditures for youth with and without dka. we used the same approach to estimate mean medical expenditures for youth by severe hypoglycemia status and predicted excess expenditure associated with severe hypoglycemia. we used the 2007 marketscan commercial claims and encounters (cce) database (marketscan database ; thompson medstat, ann arbor, mi). this database, which has been used extensively in studies of health care costs, including studies of diabetes costs (12,1416), contains fully adjudicated and paid claims for several million people with employer - sponsored health plans, including employees, spouses, and dependents (17). the cce database includes patient - level data on inpatient, outpatient, and drug claims that can be linked using encrypted enrollee information. types of health plans from which enrollees obtain services can be divided into fee - for - service (ffs) plans and fully or partially capitated plans. ffs plans include preferred provider organization (ppo) plans, exclusive provider organization plans, point - of - service (pos) plans, consumer - directed health plans, and indemnity plans. fully or partially capitated plans include hmo plans and capitated pos plans (18). cost estimation for those enrolled in ffs health plans for those enrolled in capitated plans, payment records often reflect encounter status and not the actual payment (18). youth who had health care service and prescription drug coverage through an ffs plan between 1 january and 30 december 2007. following criteria used in a previous study (19), enrollees were identified as having diabetes if during calendar year 2007 they had at least one claim for diabetes - related drugs and supplies and at least two outpatient encounters 30 days apart in which diabetes was a primary or secondary diagnosis or at least one inpatient admission with diabetes as a primary or secondary diagnosis. the requirement of at least two outpatient encounters excludes those who were misdiagnosed at the first encounter but later determined not to have diabetes (16). we used the icd-9-cm codes 250.00250.93, 357.2, 362.0362.02, and 366.41 (18) as diabetes indicators in the outpatient and inpatient claims. in the second step, youth with dm were identified to have itdm if they had at least one drug claim for insulin as indicated by therapeutic class code 172. last, youth with itdm were identified to have dka if they had any claims coded with icd-9-cm code 250.1 (2) and with severe hypoglycemia if they had any claims coded with icd-9-cm codes 251.0, 251.1, 251.2, or 250.8 (12). to determine the extent to which the number of episodes of dka or severe hypoglycemia was associated with excess expenditures, we dichotomized youth with these complications into those having one episode and those having more than one episode. we considered an individual to have had one episode of dka if they received one service with a dka code during the study period and to have had more than one episode if they had received at least two such services at least 30 days apart. we excluded 39 with missing census region or urbanity of residence (urban versus rural). we also excluded 129 people with uncommon chronic conditions (congenital heart failure, hemiplegia, lymphoma, down 's syndrome, autism, leukemia, liver diseases, and congenital heart defects). we could not consider these conditions because the number of youth experiencing each condition was very small (e.g., 10 among dka or severe hypoglycemia youth). because of its relative high prevalence and possible interaction with diabetes expenditures, we did not exclude 309 (4.1%) youth with asthma. we used stata version 10.1 (stata, college station, tx) for all analyses. we estimated four expenditure models (outpatient, inpatient, drug, and total). the presence or absence of dka or severe hypoglycemia covariates included age, sex, census region (midwest, south, west, and northeast), residence urbanity, health benefit plan type (ppo versus non - ppo), and presence of asthma. we compared the unadjusted means of the characteristics of youth by their dka or severe hypoglycemia status with student t tests. we used a generalized linear model with log link and gamma distribution to model total, outpatient, and drug expenditures because all youth had positive expenditures and expenditures were highly skewed and right tailed (20). we used a two - part model to model inpatient expenditures to account for those with no inpatient expenditures (21,22). in the first part of the two - part model, we used logistic regression to estimate the probability that someone would have had any inpatient expenditures, and, in the second part, we used a generalized linear model with log link and gamma distribution to estimate the inpatient expenditures for those who had such expenditures. we calculated model - based predicted marginal medical expenditures by dka status. in this approach, predictions were made for all observations assuming no dka status for all observations and then again assuming dka. the predicted excess expenditure associated with dka was calculated as the difference between the predicted expenditures for youth with and without dka. we used the same approach to estimate mean medical expenditures for youth by severe hypoglycemia status and predicted excess expenditure associated with severe hypoglycemia. of 7,556 youth with itdm, 1,126 (14.9%) experienced at least one dka episode. of those, 600 (53.3%) experienced one episode and 526 (46.7%) experienced more than one (mean 2.6). of all youth with itdm, 595 (7.9%) experienced at least one severe hypoglycemic episode. of those, 400 (67.2%) had one episode and 195 (32.8%) had more than one (mean 3.5). the mean age was 13 years, and those who experienced dka were significantly younger than those who did not (p 40% of the total medical expenditures incurred by youth who had dka, but excess inpatient expenditures were > 90% of the excess expenditure attributable to dka. in contrast, mean prescription drug costs were lower among youth who had dka than those who did not (p 90% of the excess medical expenditures associated with both one and more than one episode of dka. the mean annual total medical expenditure for those who had dka was 1.7 times greater than for those without and was even greater for those with multiple dka episodes. $) in 2007 for u.s. youth with itdm, by dka and severe hypoglycemia status data are means bootstrap ses with 100 replications. excess = the difference between mean medical expenditures for youth with complications and those for youth with no complications. covariates included in the model are age, sex, census regions, urbanity of residence, health plan, and asthma. the amount of all the excess expenditures was statistically significant (p 40% of the total medical expenditures incurred by youth who had dka, but excess inpatient expenditures were > 90% of the excess expenditure attributable to dka. in contrast, mean prescription drug costs were lower among youth who had dka than those who did not (p 90% of the excess medical expenditures associated with both one and more than one episode of dka. the mean annual total medical expenditure for those who had dka was 1.7 times greater than for those without and was even greater for those with multiple dka episodes. predicted mean annual medical expenditures (u.s. $) in 2007 for u.s. youth with itdm, by dka and severe hypoglycemia status data are means bootstrap ses with 100 replications. excess = the difference between mean medical expenditures for youth with complications and those for youth with no complications. covariates included in the model are age, sex, census regions, urbanity of residence, health plan, and asthma. the amount of all the excess expenditures was statistically significant (p 90% of the total excess medical expenditures attributed to dka. thus, dka was treated primarily in hospital settings. in comparison, total excess expenditures associated with severe hypoglycemia were fairly evenly divided between excess inpatient and outpatient expenditures (46.7 and 42.4%, respectively), indicating that severe hypoglycemia was treated on both an out- and inpatient basis. differences in treatment settings likely reflect differences in the complications ' nature and complexity. although more health care resources were spent to care for youth who had more than one episode of dka or severe hypoglycemia than for those who had one episode, the average excess expenditure per episode of dka or severe hypoglycemia was higher among youth who had only one episode. one somewhat unexpected finding was that expenditures for prescription drugs were less among youth who experienced dka than among those who did not. one plausible explanation is that diabetic youth who use an inadequate quantity of insulin might be at higher risk of dka. alternately, dka episodes among youth with itdm might have occurred at diabetes onset when they were still secreting some endogenous insulin and thus required less exogenous insulin. data limitations prevented us from differentiating dka episodes occurring at the onset of diabetes from those among people with an established diabetes diagnosis. our estimate of mean total medical expenditure associated with dka ($ 5,837) was less than half than that reported for adults ($ 13,046 [2007 dollars ]) with type 1 diabetes (11). the higher medical expenditures for adult diabetic patients with dka could be attributed to disease severity. for instance, the mean length of hospital stay among dka patients in our youth sample was 1.6 days, about one - fourth of adult hospital stays (6.6 days) (9). the presence of other comorbidities, such as cardiovascular diseases and chronic diabetes complications among adults, could contribute to the severity of dka in adults. our $ 3,880 estimate of excess annual expenditures associated with severe hypoglycemia was slightly lower than an estimate of excess annual expenditures associated with severe hypoglycemia among insured employees with ffs and capitated health plans in 19992000 ($ 4,355 [2007 dollars ]) (12) and a corresponding estimate among patients under managed care health plans in southern u.s. states in 2002 ($ 4,319 [2007 dollars ]) (13). the higher medical expenditure associated with severe hypoglycemia among adults could be associated with the severity of disease and chronic diabetes complications. our population was a convenience sample of youth with insurance coverage through large employer - sponsored health care programs. those in our study were likely to have a better access to health care than those uninsured or covered under medicaid. our estimates pertain to youth enrolled in ffs plans without uncommon chronic conditions. in sensitivity analyses that included these conditions, medical expenditures associated with dka or severe hypoglycemia changed significantly (results not reported). in addition, our estimates do not reflect medical expenditures for youth insured under capitated plans. finally, because we restricted our analysis to medical expenditures from administrative claims data, we did not capture the total cost of dka or severe hypoglycemia, including the cost of medical care not paid for by health insurance plans, other out - of - pocket costs, the cost of care provided by family members and by schools, and human capital losses. to the best of our knowledge, this was the first study of medical expenditures associated with acute diabetes complications among a large sample of privately insured u.s. our estimates may be useful in evaluating the economics of pre- and postdiagnostic diabetes interventions that reduce dka and severe hypoglycemia in u.s. | objectiveto estimate medical expenditures attributable to diabetes ketoacidosis (dka) and severe hypoglycemia among privately insured insulin - treated u.s. youth with diabetes.research design and methodswe analyzed the insurance claims of 7,556 youth, age 19 years, with insulin - treated diabetes. the youth were continuously enrolled in fee - for - service health plans, and claims were obtained from the 2007 u.s. marketscan commercial claims and encounter database. we used regression models to estimate total medical expenditures and their subcomponents : outpatient, inpatient, and drug expenditures. the excess expenditures associated with dka and severe hypoglycemia were estimated as the difference between predicted medical expenditures for youth who did / did not experience either dka or severe hypoglycemia.resultsfor youth with and without dka, respectively, predicted mean annual total medical expenditures were $ 14,236 and $ 8,398 (an excess of $ 5,837 for those with dka). the excess was statistically greater for those with one or more episodes of dka ($ 8,455) than among those with only one episode ($ 3,554). predicted mean annual total medical expenditures were $ 12,850 and $ 8,970 for youth with and without severe hypoglycemia, respectively (an excess of $ 3,880 for those with severe hypoglycemia). the excess was greater among those with one or more episodes ($ 5,929) than among those with only one ($ 2,888).conclusionsmedical expenditures for potentially preventable dka and severe hypoglycemia in u.s. youth with insulin - treated diabetes are substantial. improving the quality of care for these youth to prevent the development of these two complications could avert substantial u.s. health care expenditures. |
a 64-year - old woman came to our pain clinic with the symptom of right lateral side thigh pain which occurred from one year ago. her pain started from knee lateral side and the pain was radiated to buttock area. when she started to walk, the pain was triggered and whenever symptoms aggravated, she could feel the tingling sensation through a whole leg which made her limping. she described her pain as pin pricking or stabbing with her pain severitiy 7 - 8/10 on the visual analogue scale (vas). before she felt the lateral leg pain however, we did not think it is the main problem causing the symptom. although her pain always started from knee lateral side, ultimately she could feel severe pain coming from entire thigh. therefore, we performed straight leg raising and patrick test to rule out any spine and pelvic problem. she was negative to all these tests and did not show any local tenderness around back or buttock. however, we could check significant local tenderness on right lateral side of knee. also we performed knee instability test on both side which showed no abnormality. as the pain always started from the knee and aggravated by walking, we suspected some pathologic changes from inside the knee like meniscus or cruciate ligament. we decided to evaluate knee magnetic resonance imaging (mri) study and simple x - ray. her mri study showed slight degenerative change on meniscus which seemed to be unrelated to the current pain. however, we could identify high signal intensity appearing at itb through coronal and axial image (fig. 1). we assessed that itb syndrome is causing the pain. to alleviate her symptom, we prescribed 37.5 mg tramadol/375 mg acetaminophen combination tablet (paramacet tab : donga, seoul, korea), 5 mg amitriptyline per os twice daily but had little effect. we educated her about the itb specific stretching exercise which was known to improve the symptom by lengthening of the itb. she continued to perform this stretching exercise for a month but the pain relief was not satisfactory. to improve her symptom, we planned local corticosteroid injection by ultrasound guidance. we explained about the procedure, efficacy, and possible side effects of local corticosteroid injection. the skin was aseptically draped with betadine and the ultrasound transducer was covered with a sterile transparent sheath and aseptic gel. we used a 10 - 12 mhz linear transducer (iu 22, philips, netherlands). first, we marked the maximal tenderness area and the transducer was applied longitudinally to the lateral side of the knee. we could easily identify the itb, femoral lateral condyle, tibia, and recess. we targeted the needle tip beneath the iliotibial band and confirmed its location by injecting small amount of saline (fig. after one week, the patient was followed up and she was fully recovered from previous pain. one month later, she was followed up again and she could not feel any pain around knee and thigh. itb syndrome causes pain in the area of the lateral femoral epicondyle or slightly below to it, which occurs after repetitive flexion and extension of the knee, typically in a runner, cyclist, or other athlete. an important finding of physical examination is local tenderness of the lateral knee below to the epicondyle and superior to the joint line. the noble test is performed with the patient lying supine ; beginning with the affected knee flexed at 90, the leg is extended with direct pressure over the lateral femoral epicondyle, with reproducible pain near 30 of knee flexion. the ober test for distensibility of the itb is most commonly used to assess tightness of the itb. the thomas test is used to check the tightness of iliopsoas muscle, rectus femoris muscle, and itb. the differential diagnosis includes lateral meniscus tear, lateral compartment degenerative joint disease, biceps femoris tendinopathy, stress fracture, patellofemoral syndrome and lateral collateral ligament pathology. most of patients diagnosed as itb syndrome typically present symptoms localized to lateral side of the knee. however, the pain is not always localized to the lateral side of knee in which case our patient has shown, therefore, the diagnosis can be challenging in some cases. before she came to our pain clinic, she visited several other local clinics and has been heard that the thigh pain is coming from the spine disease like disc herniation or spinal stenosis. as the thigh pain is aggravated by walking or repetitive exercice, it can be considered as neurogenic intermittent claudication which is observed in spinal stenosis. the patients of itb syndrome can possibly complain thigh pain when we think the anatomical orientation of itb. the itb is formed proximally at the level of the greater trochanter as a coalescence of the tensor fascia latae and the gluteus maximus and medius muscles. anatomic reports have demonstrated that the itb is a dense fibrous connective tissue that passes distally along the thigh [7 - 9 ]. proximal to the knee joint, the itb has attachments to the intermuscular septum and the supracondylar tubercle of the femur. it continues inferiorly to insert on the gerdy tubercle at the anterolateral side of the tibia. the contact is formed at the level of the lateral femoral condyle between the itb and the underlying epicondyle and origin of the lateral collateral ligament, helping provide lateral stability to the knee joint. most of itb syndrome is a nontraumatic overuse injury which is caused by friction and rubbing of the distal portion of the itb over the lateral femoral epicondyle. orchard. proposed that an impingement zone occurs near 30 of knee flexion during foot strike and early stance phase. the itb passes over and posterior to the lateral femoral epicondyle at approximately 30 and greater of knee flexion. while this impingement happens, eccentric contraction of the tensor fascia lata and gluteus maximus is formed to exert great tension through the itb. the pathogenesis of itb syndrome involves inflammatory process and irritation of the posterior fibers of itb and lateral synovial recess. in cases of refractory itb syndrome itb syndrome can be diagnosed with the appearance of high - intensity signal on t2 weighted image which appears over the lateral epicondyle deep to the itb, and marked thickening of the distal itb. ultrasonography can also be used as a diagnostic tool and it demonstrates thickening of itb in case of patients. monitoring the thickness of itb might be a useful variable in the diagnosis of itb syndrome, moreover, to evaluate the evolution of this condition. oral nonsteroidal anti - inflammatory drugs and corticosteroid injections can be used to reduce the acute inflammatory response. gunter and schwellnus reported that local corticosteroid infiltration around itb (1 ml 1% lidocaine plus 40 mg methylprednisolone) compared with control group effectively decreased pain during running in the first two weeks of treatment in patients with recent onset itb syndrome.. therefore, their report does not suggest any ideal location for corticosteroid injection. for symptom improvement of our patient the main benefit of ultrasound guidance during corticosteroid injection is the ability to identify vascular structures and nerves located in the needle pathway in order to avoid these structures and be sure to inject the medication into the most optimal location. ultrasonography enables real time examination, which promotes dynamic evaluation of various tendons, and continuous monitoring during any type of injection. in contrast to the fluoroscopy, ultrasound does not produce any harmful reaction to both physician and patient. moreover, ultrasound guided accurate injection can result in significantly greater clinical improvement in pain and function over blind (landmark guided) injections. for corticosteroid injection, initially, we identified several anatomical structure including the itb, lateral synovial recess and femoral epidcondyle. we targeted the potential space between the itb and overlying lateral femoral epicondyle because most of inflammatory and irritation process occurs here. the injection was successfully performed at this site without any event and the patient showed excellent recovery from pain. if the ultrasound guided corticosteroid injection of itb should be performed, we suggest that our method can be a useful option. | a 64-year - old woman visited our pain clinic with the pain of right lateral side of thigh for one year. her pain always started from knee and was radiated to buttock area when symptom was severe. she showed significant tenderness at knee lateral side and local tightness at lateral thigh. magnetic resonance image of the knee was performed and we could identify high signal intensity of iliotibial band through coronal and axial view. in spite of medication and physical stretching exercise of iliotibial band for one month, she did not show any improvement of pain. to alleviate her symptom, ultrasound guided local corticosteroid injection targeting beneath the iliotibial band was performed. after the procedure, the reduction of pain was significant and there was no need for further management. |
the internet is increasingly utilized in place of traditional textbooks to obtain medical information in a variety of disciplines.16 physicians access the internet for a range of purposes, including resident education, communication of information to peers, research, and as a clinical decision - making tool. both the medical literature and anecdotal experience suggest that radiologists, in particular, are increasingly using the internet and internet - based search engines. as early as 1999, a survey of 210 european radiologists and radiology residents reported that 18% access the internet daily for radiologic activities. a total of 69% of all respondents had used the internet at some time for literature searching and 12% at some time for teaching or education. the study further found that 28% of respondents had accessed electronic journals.4 a 2002 survey of 2,200 physicians found that 65% used the internet for literature searching, 53% used it to search for medical information, and 45% accessed online journals.5 this use has increased significantly in the past decade. in a 2005 survey of 92 radiologists, 97% reported using the internet for radiology education, and 69% found the reliability of the information to be equal to that of information from traditional sources.6 the same survey found that 31% of respondents used the google search engine most frequently to find radiologic information. physicians find the internet to be a reliable source of medical information that can be directly applied to clinical problems. reports have documented the widespread belief among physicians that medical content on the internet has professional value and that better care can be provided using this information.5 a recent study described the use of the google search engine as a diagnostic tool to make the correct diagnosis in 58% (15) of the 26 diagnostic cases published in the case records of the new england journal of medicine in 2005.7 search engines help physicians formulate differential diagnoses and consequently provide better clinical care, especially in difficult cases. google, yahoo, and numerous other generic search engines can be used to provide radiologists with information. however, these generic search engines are designed to provide general results for all users rather than results tailored to the interests of a specific group of users. use of these generic search engines requires radiologists to sift through a substantial volume of non - radiology material and irrelevant links in order to arrive at relevant information. consequently, using generic search engines to obtain radiologic information may not provide results that are sufficiently focused and efficient for point - of - care clinical decision making, education, and research. these search engines utilize various algorithms to narrow the scope of their results to links that are relevant to the radiologist. yottalook.com is a free search tool developed by radiologists to make web searches more efficient for medical imaging professionals. this radiology - centric vertical search engine optimizes searches to yield results that are relevant to medical imaging. it then indexes the key concepts, extracts medical images, and identifies semantic relationships within a website. these relationships are then used by its search algorithm for image retrieval and are also applied to a google custom search to provide results that are optimized for clinical and research purposes. when one initiates a search, yottalook applies a natural language query analysis to analyze the search term to better understand the purpose of the user s search. it then applies a semantic ontology that incorporates radlex to expand the user s query by adding synonyms, parent and child terms, and other related terminology.8 yottalook also applies its query expansion technology to its google books site to provide full - text search capability within medical imaging textbooks. finally, it offers users yottalinks that provide high - yield editorial content based on the specific search term entered.9 we describe searches performed using the website www.yottalook.com over a 3-week period, 7 months after its initial launch. for every 25th consecutive yottalook query from august 1, 2007 to august 24, 2007, the time of search, ip address, and other data were downloaded for retrospective analysis. institutional review board (irb) approval was not required according to our local irb. two authors (res, mjs) sorted the terms into predefined categories including 11 radiology subspecialties (musculoskeletal, pulmonary, gastrointestinal, genitourinary, neuroradiology, vascular and interventional, nuclear, ultrasonography, pediatrics, breast, and cardiac) and three other categories (general radiology, other medical conditions, and other queries). radiology subspecialty allocation was determined through consensus using criteria defined by the american board of radiology in its diagnostic radiology study guide.10 reviewers utilized radiology texts and medline searches of the primary medical literature to assist in categorization of searches. search terms that met criteria for inclusion in more than one radiology subspecialty were classified into multiple subspecialties. radiology subspecialties were defined as follows : musculoskeletal radiology included all imaging modalities related to normal physiology or pathology of the musculoskeletal system, including disorders of the bone marrow. pulmonary radiology included diseases of the lungs, pleura, mediastinum, and all of its structures excluding the heart and great vessels. gastrointestinal radiology included imaging of the esophagus, stomach, small and large intestines, biliary tract, liver, spleen, pancreas, peritoneal cavity, and abdominal wall. genitourinary radiology included searches of the kidneys, adrenal glands, ureter, bladder, urethra, and the male and female genital systems. neuroradiology was defined as imaging related to the skull, sinuses, mastoids, spine, head and neck, thyroid, parathyroid, and pituitary glands. vascular and interventional radiology included all imaging of the arteries, veins, and lymphatic structures. ultrasonography included use of the modality to investigate the head and neck, thorax, abdomen, pelvis, extremities, breast, scrotum, vascular system, uterus, and fetus. the category of general radiology included searches that were radiologic in nature but did not fit into established radiology subspecialties. terms that did not fit into a specific subspecialty and were medical rather than radiological in nature were classified as other medical topics. terms not fitting into any of the above - described categories were classified as other queries. international whois databases were used to determine the search origin of queries from their internet protocol (ip) address. this location was also used to categorize queries into geographical regions using accepted classification systems. geographic regions included africa, asia, europe, north america, oceania, and south america. after the search origin location was identified, the time of the search was converted to that region s local standard time to analyze searches by time of day. the presence of abbreviations and the language of the search were also noted for each search term. data analysis was performed using microsoft excel and accepted methods for calculating 95% confidence intervals for proportions with nominal data. during the study period, 25,075 queries were placed at www.yottalook.com, and 1,005 terms were retrospectively categorized in this study. nine hundred sixty (95.5%) searches were classified into a single radiology subspecialty, 44 (4.4%) terms were classified into two subspecialties, and one (0.10%) term was classified into three subspecialties. neuroradiology was searched significantly less frequently than musculoskeletal imaging and searched significantly more frequently than the other remaining subspecialties. magnetic resonance imaging (mri) was indicated in 42 (4.2%) searches, and computed tomography was indicated in 41 (4.1%). fig 2search query analysis by reference to imaging modality presented as modality, number of times mentioned in search queries, and as percent of overall queries. search query analysis by reference to imaging modality presented as modality, number of times mentioned in search queries, and as percent of overall queries. searches were placed from all continents (table 1). the largest number (793, 78.9%) of searches originated from north america. six queries (0.6%) had ip addresses that could not be traced to their origins using whois software. six hundred seventeen (61.4%) searches were executed between 9 a.m. and 5 p.m. at their local points of origin, whereas only 62 (6.2%) were executed between 12 a.m. and 7 a.m. (fig. abbreviations were used in 186 (18.5%) searches, and 93 (9.3%) searches contained misspellings. fig 3search engine utilization by local standard time.table 1search - term categorization by geographic regionsearch term categoriesgeographic regionnorth americasouth and central americaeuropeoceaniaasiaafricaqueriespercent totalqueriespercent totalqueriespercent totalqueriespercent totalqueriespercent totalqueriespercent totalmusculoskeletal19024.02123.62027.4939.100.000.0pulmonary556.966.745.528.716.3240.0gastrointestinal749.31415.7912.314.3212.500.0genitourinary729.189.079.6313.0212.500.0neuroradiology14818.71213.51216.4521.7425.000.0vascular and interventional617.71112.434.114.316.3120.0nuclear182.300.011.400.000.000.0ultrasonography172.133.434.100.016.300.0pediatrics151.911.100.000.016.300.0breast131.633.400.000.016.300.0cardiac486.133.411.428.716.3120.0general radiology334.211.1912.300.016.3120.0other medical topic202.522.222.700.000.000.0other query293.744.522.700.016.300.0total searches by origin79378.9898.9737.3232.3161.650.49 search engine utilization by local standard time. more than half of all internet searches on www.yottalook.com came from three of the radiologic subspecialties : musculoskeletal radiology, neuroradiology, and gastrointestinal radiology. the least frequently searched categories included breast imaging, nuclear imaging, and pediatric radiology, which when combined accounted for approximately 10% of all search queries. statistically significant variation was noted in the frequencies of search queries for various radiologic subspecialties. radiology - specific search engines can be used for many purposes, including clinical, research, and educational applications, and we would anticipate different results for each of these purposes. it is not surprising that musculoskeletal imaging and neuroradiology were searched most frequently. these areas are generally considered to represent the bread and butter applications of mri which was the most frequently referenced imaging modality. the least frequently searched categories of nuclear medicine, pediatric, and breast imaging may represent relatively subspecialized areas that may not be as commonly searched by general radiologists. additional analysis should be performed to better understand the variability in queries among the different radiology subspecialties. this regional variation may result from variability in the availability of specific modalities and the preferences of practicing physicians and radiologists for various modalities, as well as differences in the prevalence of diseases in these regions. our data suggest that it may be advantageous to include regional differences in the development of algorithms designed to optimize the effectiveness of these search engines. radiology - specific search engines are used globally to collect information to investigate all radiologic modalities, especially during the typical workday. the nature of searches occurring 24 h / day in adjusted local times may suggest that search engines are being used as real - time clinical decision - making tools, particularly for mr and computed tomography questions. late - night and early morning searches may be originating from on - call attending radiologists, radiology residents, and teleradiologists. frequent natural language abbreviations and misspellings suggest that search engines should be prepared to interpret and appropriately modify common searches for increased user efficiency. the limitations of our methodology include the inability to precisely assess the level of expertise and the professional identity of persons using the search engine. the majority of searches, however, were relatively homogenous. that is, most were performed during the regular business day and a few searches were categorized as therefore, we believe it is likely that most searches included in this study were placed by general radiologists, subspecialty radiologists, radiology residents, students, and other physicians or other radiology professionals we also have limited data from which to assess the practice and search patterns in asia and africa because of the smaller number of total searches from these regions. another limitation of the study is the fact that it focused on the use of the search engine soon after its initial release. a follow - up analysis would be useful, especially because utilization of the search engine has increased greatly over the past several months. radiologic searches using a radiology - specific search engine are more frequently undertaken within some radiology subspecialties than in others. radiologic searches with a radiology - specific search engine vary by subspecialty and modality searched, geographic region of search origin and time of day at which the search was conducted. this variability may reflect the relative caseload of radiologists as well as dispersion and utilization trends of various imaging technologies. this study demonstrates varying international utilization of specialized internet search engines across all imaging modalities. we believe current and future internet - based search engines should take this geographic variation into account in determining their search methodologies to provide optimal and efficient search results for practicing, researching, and teaching radiologists. further refinements could provide results optimized to the type of user initiating the search or could even utilize the history of a user s previous searches to provide more | internet - based search engines have become a significant component of medical practice. physicians increasingly rely on information available from search engines as a means to improve patient care, provide better education, and enhance research. specialized search engines have emerged to more efficiently meet the needs of physicians. details about the ways in which radiologists utilize search engines have not been documented. the authors categorized every 25th search query in a radiology - centric vertical search engine by radiologic subspecialty, imaging modality, geographic location of access, time of day, use of abbreviations, misspellings, and search language. musculoskeletal and neurologic imagings were the most frequently searched subspecialties. the least frequently searched were breast imaging, pediatric imaging, and nuclear medicine. magnetic resonance imaging and computed tomography were the most frequently searched modalities. a majority of searches were initiated in north america, but all continents were represented. searches occurred 24 h / day in converted local times, with a majority occurring during the normal business day. misspellings and abbreviations were common. almost all searches were performed in english. search engine utilization trends are likely to mirror trends in diagnostic imaging in the region from which searches originate. internet searching appears to function as a real - time clinical decision - making tool, a research tool, and an educational resource. a more thorough understanding of search utilization patterns can be obtained by analyzing phrases as actually entered as well as the geographic location and time of origination. this knowledge may contribute to the development of more efficient and personalized search engines. |
coronary artery ectasia (cae) has been defined as localized or diffuse dilation of the coronary arteries exceeding the 1.5 fold of normal adjacent segment in coronary angiography [1, 2 ]. cae is a rare finding among coronary artery anomalies and considered to be of either congenital or acquired origin [2, 3 ]. it is estimated that 50% of cae is related to atherosclerosis, whereas 20%30% of cases may be due to congenital anomalies [3, 5 ]. although it has been suggested that ectasia is commonly a variant of atherosclerosis, it is not known clearly why some patients with obstructive coronary artery disease (cad) develop cae, whereas others do not [13 ]. tumor necrosis factor - alpha (tnf-) and interleukin-6 (il-6) are significantly associated with coronary atherosclerosis [8, 9 ]. although the increase in the plasma levels of tnf- and il-6 in obstructive cad is well known, limited data are available in cae. in this study, we aimed to evaluate the plasma levels of the cytokines ; tnf- and il-6 in cae patients. the study population was selected from a series of 1820 consecutive patients who underwent coronary angiography in our hospital between january 2008 and september 2008 due to the presence of chest pain or positive or equivocal results of noninvasive screening tests for myocardial ischemia. out of the 1820 patients, 36 consecutive patients who had angiographically normal coronary arteries with cae (group i, 28 male, mean age : 58.2 12 years) and accepted to participate to our study after giving informed consent were identified and compared with age, and sex - matched 32 consecutive participants with angiographically show normal coronary arteries without cae (group ii, 25 male, mean age : 57.2 10 years). no significant difference was present between the two groups regarding the use of acetylsalicylic acid, beta blockers, nitrates, calcium - channel blockers, and statins. the study protocol was approved by the institutional ethics committee, and the study was conducted in accordance with the decleration of helsinki and good clinical practice (gcp)/international conference on harmonization (ich) guidelines. subjects were excluded if they had evidence of traumatic injury, acute coronary syndrome history, obstructive cad, left ventricular dysfunction, left ventricular hypertrophy, cardiomyopathies, congenital heart disease, valvular heart disease, any abnormality in thyroid function test, arrhythmias, clinically unstable medical illness such as hepatic or renal impairment ; a history of seizure, head trauma, or stroke, active infection, allergy, active inflammation, collagen vascular disease, cancer, and any other primary disease interfering with immune functions. in all patients, coronary angiography was performed using the philips angioscop x - ray (integris bh 5000, philips medical systems, best, the netherlands) using standard judkins technique. two experienced cardiologists, unaware of the patients clinical characteristics and biochemical results, reviewed all of the angiographic images. we used iohexol (omnipaque, nycomed ireland, cork, ireland) as contrast agent during coronary angiography in the study patients. a coronary diameter index was defined for each segment as the coronary diameter divided by the body surface area (bsa). a coronary segment with a diameter index 1.5 fold of the control group was defined as ectatic. when there was no identifiable adjacent normal segment, the mean diameter of the corresponding coronary segment in the control group served as normal values. blood samples of all individuals were taken 24 hours after coronary angiography from an antecubital vein. blood samples were centrifuged immediately at 250 g for 10 minutes and stored at + 4c until assay. triglyceride, total cholesterol, low - density lipoprotein (ldl) cholesterol, and high - density lipoprotein cholesterol concentrations were measured by automated chemistry analyzer (aeroset, abbott, ill, usa) by using commercially available kits. ten ml of heparinized venous blood were collected with the plastic tubes from subjects at 08.00 a.m. blood samples were santrifuged at 3000 rpm (rotor diameter : 16 cm) and preserved at 80c. analyses were performed by the immunologists, who were blinded to the condition of the samples. il-6, tnf- enzyme - linked immunosorbent assay (elisa) kits were purchased by biosource international inc. (camarillo, calif, usa) and used according to the recommendations of the manufacturer. the minimum detectable doses of tnf- and il-6 are 1.1 pg / ml, 2.2 pg / ml, respectively. statistical analysis was performed by using spss for windows 11.0 (chicago, ill, usa). continuous variables were expressed as mean sd, and categorical variables were expressed as percentage. comparison of categorical and continuous variables between groups was performed by using x test and student 's t - test or mann - whitney u - test. the correlation between tnf- and il-6 levels and the number of ectasic vessels was assessed by the pearson correlation, or the spearman tests were appropriate reproducibility was estimated by analyzing all recordings on 2 separate occasions (intraobserver variability) ; to assess interobserver variability, investigators were blinded. baseline demographic, clinical, and angiographic characteristics of the two groups were similar (table 1). tnf- and il-6 levels were significantly higher in cae group than controls (15.6 11.2 pg / ml versus 7.8 3.7 pg / ml, p <.001, and 17.2 12.6 versus 7.6 2.1 p <.0001, resp.) (table 2). besides no correlation was found between the tnf-, il-6 levels and the number of ectatic vessels (r = 0.012, and r = 0.138, resp. the main finding of the present study was that patients with isolated cae had significantly higher tnf - alfa and il-6 levels compared to control subjects with angiographically normal coronary arteries. cae is a well - recognized angiographic finding of abnormal coronary dilatation, and detected in 0.3%5.3% of consecutive angiographic studies. the gold standard for diagnosis of this type of ectasia is coronary angiography, which provides information about the possible size, sample, location, and number of ectasias. clinically, it predisposes to adverse coronary events like vasospasm, thrombosis, and dissection. in some studies, although many etiologies of cae have been reported, the common coexistence with cad has raised the idea that cae may be a variant of cad. individual case reports have shown that isolated cae may be a cause of silent myocardial ischemia. in this study, we found significantly higher levels of il-6 in cae patients when compared to control subjects with normal coronary arteries. we suggest that this inflammatory state in the cae patiens is responsible for higher tnf- and il-6 levels in cae. a recently published investigation reported that coronary flow reserve is significantly reduced in patients with cae and put forward that microvascular dysfunction may be the underlying cause of exercise - induced myocardial ischemia. there is now a well - known common aggreement that atherosclerosis is an inflammatory disorder of the vessels. although cause and pathways of atherosclerotic process are probably multiple and different in various clinical settings, the data showed that an inflammatory process was involved in all stages of atherosclerosis including coronary spasm, delayed coronary flow, coronary microvessel dysfunction, silent myocardial ischemia, and restenotic process [16, 17 ]. as a results of these data, the idea of chronic inflammation as one of the key factors involved in atherosclerosis has come out and broaden into a new area of atherosclerotic diseases. a few recent data have also emphasized that cae is associated with systemic inflammatory response exposed by inreased inflammatory cytokines and c - reactive protein (crp). recently, a study indicated a cytokine - induced tissue inflammation in the pathogenesis of abdominal aortic aneurysms, and it has been documented that circulating il-6 levels increase in these patients. studied with cae, and found that serum il-6 levels were significantly higher in patients with cae compared to controls. studied the cae and found that mean fluorescence intensity of cd45 and cd11b on the monocyte surface of patients with cae was significantly higher when compared to controls. they speculated that increased levels of cellular adhesion molecules in patients with cae may be an indicator of endothelial activation and inflammation and are likely to be in the causal pathway leading to coronary artery ectasia.. demonstrated that the levels of crp, a specific marker of chronic inflammation, were significantly higher in patients with isolated cae. a recently published study showed that patients with isolated cae have raised levels of plasma soluble intercellular adhesion molecule-1 (icam-1), vascular cell adhesion molecule-1 (vcam-1), and e - selectin in comparison with patients with obstructive cad without cae and subjects with normal coronary arteries. in addition, turhan. evaluated crp levels as a specific marker of inflammation in patients with cae. all these data indicate the presence of a chronic inflammation in the coronary circulation of the patients with cae. accordingly, cae has been suggested to be a destructive inflammatory lesion of the vascular wall. briefly, the approving of the importance of inflammation in atherosclerosis has both theoretical and practical clinical implications. the data have demonstrated that cae is associated with inflammatory response exposed by increased inflammatory cytokines. in conclusion, we found that tnf- and il-6 levels were significantly higher in cae patients than subjects with normal coronary arteries. we think that tnf- and il-6 measurements may be a good prognostic value in cae patients as in stenotic ones, suggesting that vascular wall inflammation may play a role in the pathogenesis of cae. further placebo - controlled studies are needed to evaluate the clinical significance of increases in these markers. | background / aim. coronary artery ectasia (cae) is considered as a variant of atherosclerosis. tumor necrosis factor - alpha (tnf-) and interleukin-6 (il-6) are among the sensitive markers of systemic inflammation. the aim of this study was to evaluate the plasma levels of the cytokines ; tnf- and il-6 in cae patients. methods. plasma concentrations of tnf- and il-6 were measured in 36 patients with cae (28 males, mean age : 58.2 12 years), and results were compared with age and sex - matched controls (n = 32) without coronary artery ectasia. tnf- and il-6 concentrations in blood were assesed by enzyme - linked immunosorbent assay (elisa). results. baseline characteristics of the two groups were similar. tnf- and il-6 levels were significantly higher in cae group than controls (15.6 11.2 pg / ml versus 7.8 3.7 pg / ml, p <.001, and 17.2 12.6 versus 7.6 2.1 p <.0001, resp.). conclusion. cae patients showed increases in tnf- and il-6 levels compared to the controls. this study provides evidence for alterations in the proinflamatory cytokines which suggest the involvement of the immune system in the pathophysiology of cae. further placebo - controlled studies are needed to evaluate the clinical significance of this increase in tnf- and il-6 levels. |
the pe / pe_pgrs multigene family unique to mycobacteria accounts for about 5% of the mycobacterium tuberculosis genome. the family includes 38 pe - encoding genes and 61 pe_pgrs - encoding genes scattered throughout the genome, characterized by a relatively conserved nh2-terminus of ~110 amino acids (aa) followed by a cooh - terminal glycine - rich repeat region ranging from ~100 aa to over 500 aa in length 1., 2.. several pe_pgrs proteins have been demonstrated to be associated with the replication and survival of m. tuberculosis within macrophages of infected host tissues and are important for pathogenesis (3). one of the functions proposed for these proteins is that they are a source of antigenic variability to evade host immune responses (1). other observations suggest the possibility that pe_pgrs proteins could either be cell surface constituents (adhesins) (4) that can influence bacterial cell structure (5) or could interfere with immune responses by inhibiting antigen processing (6). a recent study has shown that the evolution and expansion of the pe and ppe families is closely associated with the esat-6 (esx) gene cluster and has suggested a functional interdependence (7). as the esat-6 cluster encodes proteins involved in secretion and membrane pore formation, it is likely that the esat-6 gene cluster - encoded proteins might also be required for secretion of some of the pe family proteins. despite these reports, the precise function and underlying mechanism of action still remain unknown for this large family of proteins. the aim of this study was therefore to investigate all of the 61 pe_pgrs proteins in the pe subfamily to predict whether they carry any distinct function in m. tuberculosis. attempts were made to determine their underlying mechanism of action, and an explanation was sought for answering why there are so many different pe_pgrs proteins in m. tuberculosis. attempts to identify similar proteins in databases to provide insights into their functions have been difficult owing to the repetitive nature of these proteins. similarity searches of pe_pgrs proteins invariably identify only other pe_pgrs family members or glycine - rich proteins such as those found in highly elastic plant cell wall proteins (8). to search specifically for non - mycobacterial microbial homologues that have so far not been identified, we analyzed the microbial genome databases (http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi) excluding mycobacterium species (9). using the widely investigated pe_pgrs protein rv1818c as query and without masking the low complexity regions, one of the proteins revealed from the blast results was an rtx (repeat in toxin) toxin of magnetococcus sp. the rv1818c protein has 42% identity over 100% stretch of its pgrs domain to the glycine - rich domain of the cog2931 protein (data not shown). although this domain corresponds to only a small region (5.8%) of the large rtx protein, the fact that some pe_pgrs proteins were reported as surface - exposed adhesin - like molecules 5., 10. prompted us to examine this similarity more carefully. the family of rtx toxins includes haemolysin, cyclolysin, leukotoxin, and metallopeptidase, and these proteins seem to have two properties in common. first they bind calcium, and second they contain a multiple tandem repeat of nona - peptides, ggxgxd / nxux, where x = any amino acid and u = unpolar / large hydrophobic residue at the c - terminal end of each protein 11., 12.. it was subsequently found that these sequence repeats constitute a ca - binding structure called a parallel -helix or parallel -roll and might participate in host cell binding (13). to examine whether rv1818c also contains this nona - peptide repeat corresponding to a ca - binding motif, we searched for ggxgxd / nxux as the calcium - binding motif and detected numerous such motifs in rv1818c (figure 1). subsequently the presence of this motif was examined in all the 61 pe_pgrs proteins. among the different pe_pgrs proteins grouped according to domains (not shown), the other five pe_pgrs proteins that lacked this motif were rv0742, rv0832, rv0978c, rv3652, and rv3812. however, among the five proteins, rv0832 (137 aa) may be frame - shifted in m. tuberculosis h37rv to be fused with a protein of 749 aa encoded by rv0833 ; rv0978c is a protein of 331 aa that contains an unusually short (78 aa) prgs motif ; while rv3652 (104 aa) may also be a frame - shifted pe_pgrs protein. all these proteins belong to both classical and non - classical types of pe_pgrs proteins. the maximum number of repeats was found in rv3345c where 77 copies of this calcium - binding parallel -helix or -roll motif ggxgxd / nxux were present (table s1). a total of 911 such calcium - binding motifs, among which 403 are ggxgxdxux type and the remaining 508 are ggxgxnxux type, were detected among all the pe_pgrs family members. the number of repetitive motifs varied according to the length of the open reading frames (orfs), and the inter - motif distance was not fixed. for example, rv1818c (498 aa) contains 9 motifs and the inter - motif distance varies from a minimum of 3 aa to as high as 67 aa (figure 1). this is unlike rtx toxins where the calcium - binding motifs are not distributed so randomly. in this context, it is worth mentioning that the u residues of ggxgxd / nxux motifs in pe_pgrs proteins are not always unpolar / large hydrophobic residues in nature (table s1). in more than 70% of these cases, u = g. in the remaining cases, u could be any arbitrary residue except for cystein (data not shown). we also determined the secondary structure of all the 61 pe_pgrs proteins including rv1818c to reaffirm the presence of -strands intercepted by coils in the c - terminal pgrs domain, which is a characteristic of parallel -roll structure (data not shown). to identify structurally important folds of pe_pgrs proteins employment of the 3d - jury system (14) to predict a possible protein fold of known function, using the c - terminal pgrs domain (382 aa) of rv1818c as query sequence, revealed a ca - binding fold with a statistically moderate score (table s2). the highest fold recognition scores were compiled next for the pgrs domains of all 61 proteins (table s2). interestingly, ~70% of the total pe_pgrs proteins exhibited a common fold to the c - terminal -roll ca - binding domain of serratia marcescens metalloprotease [pdb i d : 1srp (15) and 1sat (16) ] and the alkaline protease of pseudomonas aeruginosa ifo3080 [pdb i d : 1akl (17) and 1kap (18) ], all of them belong to the rtx family. the rv3344c protein with fold prediction data greater than the confidence threshold limit set by 3d - jury system (table s2) was used subsequently to generate an optimized 3d molecular model using the alkaline protease of p. aeruginosa (pdb i d : 1akl)(17) as the template. it is important to mention here that although according to the genbank report, the rv3344c gene (accession no. yp_177961) might be a gene fragment that should be in - frame with a following orf (mtv016.45c), no frame - shift was found when checked in bac and cosmid clones (as of genbank latest update : 24-may-2007)., 19.. the predicted model (figure 2) was found to adopt ca - binding parallel -helix or parallel (-roll structures (13) located at the turn of coil regions. the models 18., 19. created for the pe_pgrs proteins with 1akl/1kap/1sat/1srp (pdb ids) fold scores greater than 25.0 were also predicted to hold calcium ions in the individual glycine - rich nona - sequence motifs (data not shown). our results presented here suggest that the highly homologous pgrs domain of the majority of the pe subfamily proteins have calcium - binding motifs and are therefore likely to be calcium - binding proteins. calcium - dependent adhesins are known to exist in the soil bacterium rhizobium species where their attachment to the developing root hairs of leguminous plants is considered to be the first step in the host - specific infection process that leads to a nitrogen - fixing symbiosis (20). analogous to the rhizobium nodulation gene nodo that encodes a ca - binding protein involved in interactions with plant root cells in a ca - dependent way, it is possible that a similar ca - dependent pe_pgrs - mediated interaction may exist in m. tuberculosis with host cells 4., 10.. the earliest interactions of m. tuberculosis with macrophages are known to result in a number of alterations in ca signaling events critical for phagosome maturation 21., 22., 23.. in macrophages, activation of cytosolic ca - regulated enzyme ca / calmodulin - dependent protein kinase ii (camkii) is essential for the phagosome - lysosome fusion (24). other studies have demonstrated that m. tuberculosis blocks this pathway via inhibition of sphingosine kinase, a macrophage enzyme that increases cytosolic ca levels 22., 25.. furthermore, the maturation and the acidification of myctobacterial phagosome could be restored by artificially raising the cytosolic ca levels using ca ionophores (21) or receptor stimulation with atp (26). this has suggested that m. tuberculosis depletes internal ca stores in infected human macrophages 21., 26.. in the light of these observations, a role for ca - binding pe_pgrs proteins could be envisaged as follows. the initial non - specific attachment of m. tuberculosis to the host alveolar macrophages via ca - dependent pe_pgrs proteins will cause a sudden dip in calcium concentration at the focal point of host pathogen interaction 27., 28.. such an event would lead to a fall in the cytosolic ca levels in macrophages, ultimately preventing phagolysosome fusion. we thus postulate that the initial host - pathogen interactions could play a very crucial role to the sensing and establishment of the bacilli s intracellular pathogenesis. the generalized calcium - dependent adhesion ability of pe_pgrs proteins does not rule out the possibility that these proteins have additional functions. existence of non - classical type of pe_pgrs proteins (data not shown) with protein folds other than conserved parallel -roll calcium - binding folds (table s2) indicates that some pe_pgrs proteins might have yet undiscovered additional virulence - related functions that help the bacilli to survive in infected host (6). additionally, it will also be of worth to see if these initial pe subfamily protein(s)-host cell interactions can promote membrane cholesterol accumulation at the site of mycobacterial entry (29) that might eventually modulate the membrane depolarization event needed for the entry of external ca. the genome information of m. tuberculosis strain h37rv was retrieved from ncbi genome database as accession nc_000962 (1). subsequently, the re - annotated m. tuberculosis genome sequence was consulted from camus. an indigenously developed algorithm was written in c++ language to extract all the 61 pe_pgrs orf sequences. to search specifically for non - mycobacterial microbial homologues of pe_pgrs proteins, we analyzed the microbial genome databases (http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi) excluding mycobacterium species (9). classification of all the 61 pe_pgrs proteins from m. tuberculosis h37rv based on domain patterns was performed using the prosite database (31) at http://au.expasy.org / prosite/. fold recognition data for these proteins were compiled using the 3d - jury system (14) available via the structure prediction meta server (http://meta.bioinfo.pl/) to predict a possible protein fold of known function in each of the pe_pgrs proteins. the model of parallel -roll structure of pgrs domain of rv3344c with potential calciums interacting with glycine - rich nona - peptide motifs was generated by insightii software (accelrys inc. nb conceived and supervised the study, collected and analyzed the data, and prepared the manuscript. bs collected the data relating to the fold, created the models, and assisted in manuscript preparation. nb conceived and supervised the study, collected and analyzed the data, and prepared the manuscript. bs collected the data relating to the fold, created the models, and assisted in manuscript preparation. | the pe_pgrs family of proteins unique to mycobacteria is demonstrated to contain multiple calcium - binding and glycine - rich sequence motifs ggxgxd / nxux. this sequence repeat constitutes a calcium - binding parallel -roll or parallel -helix structure and is found in rtx toxins secreted by many gram - negative bacteria. it is predicted that the highly homologous pe_pgrs proteins containing multiple copies of the nona - peptide motif could fold into similar calcium - binding structures. the implication of the predicted calcium - binding property of pe_pgrs proteins in the light of macrophage - pathogen interaction and pathogenesis is presented. |
the juxtaoral organ of chievitz (jooc) is a normal anatomical structure considered of neuroepithelial origin with no known function is located within the soft tissue in the buccotemporal fascia on the medial surface of the ascending ramus. jh chievitz, a danish anatomist, first described jooc in 1885 while studying human embryos. however, this structure is not only unique for adults but also has been reported in some other species and in reptiles. this enigmatic vestigial structure has been designated with various other names depending on its embryologic origin as orbital inclusions, buccopharyngeal tract, buccotemporal organ and juxtaoral organ. as a matter of fact, the only practical importance of awareness of this structure lies in the potential of being misdiagnosed as perineural invasion in a patient with oral squamous cell carcinoma, which can be one of the most treacherous pitfalls in oral pathology. hence, the basic aim of this short communication is to reveal the importance about this organ and enlighten the oral pathologist about this histopathological structure, thus preventing extensive and unnecessary investigations. he was born on october 16, 1850, in svendborg which is a town on the island of funen in south central denmark. chievitz graduated in 1869 from soro, which is a town in region sjlland on the island of zealand in east denmark. he practiced a short time before he was employed in 1877, in the anatomy under professor theodor schmidt (18251880). in 1881 he noted it in 10-week - old embryos during his study on the development of salivary glands. originally thought to be of embryonic origin, jooc starts as an epithelial thickening of the stomodeum and invaginates into the subjacent mesenchyme. this epithelial bud then detaches from the oral epithelium and becomes innervated by a buccal nerve branch receiving vascular supply from the buccal artery. the jooc measures between 7 mm and 15 mm in length and between 1 mm and 2 mm in diameter. if it is more than 10 mm in diameter, then clinicians are likely to suspect submucosal tumor or hyperplasia of jooc. microscopically, the epithelial component consists of circumscribed nests of nonkeratinizing squamous, columnar and occasionally, basaloid epithelial cells with a definite glandular or organoid pattern with no keratin formation. three concentric domains of connective tissue encase the epithelial islands as shown in figures 1 and 2. hand drawn illustration showing epithelial component with circumscribed nests of nonkeratinized squamous, columnar and basaloid epithelial cells with a definite glandular or organoid pattern with no keratin formation within loose connective tissue stroma representative of juxtaoral organ of chievitz. the juxtaoral organ of chievitz is composed of nests of epithelial parenchyma embedded in highly organized connective tissue stroma rich in nerve bundles (courtesy : jerad m gardner, md, university of arkansas for medical sciences, usa ; daifullah al aboud. 2014) courtesy : arvind venkatesh, department of oral pathology and microbiology, subharti dental college and hospital, meerut, uttar pradesh, india. well - circumscribed epithelial sprouts present within the loose connective tissue stroma representative of juxtaoral organs of chievetz (h and e, 100). inset : cells showing paler cytoplasm and clear cell - like features (h and e, 1000) juxtaoral organ of chievitz : a histopathological masquerade. indian j med paediat oncol 2015;36:193 the inner layer called stratum fibrosum internum consists of dense collagen fibers that are separated from the epithelial islands by a distinct basal laminathe middle layer, stratum nervosum, is characterized by loose connective tissue stroma, populated with myelinated and nonmyelinated fibersthe outer layer, the stratum fibrosum externum, connects to the muscle fascia of the buccotemporalis. the inner layer called stratum fibrosum internum consists of dense collagen fibers that are separated from the epithelial islands by a distinct basal lamina the middle layer, stratum nervosum, is characterized by loose connective tissue stroma, populated with myelinated and nonmyelinated fibers the outer layer, the stratum fibrosum externum, connects to the muscle fascia of the buccotemporalis. histochemically, the available ck profiles to date suggest that the epithelial nests of jooc share the immunohistochemical phenotype of nonkeratinized stratified squamous cells. alkaline phosphatase activity of the epithelial component of the jooc and a possible mechanoreceptor function due to a close approximation of jooc to structures resembling pacinian corpuscles have also been documented. jooc is an innocuous variation of normal anatomy and carries no risk for malignant transformation and no recurrence after its removal. | the juxtaoral organ of chievitz is a normal anatomical structure located within the soft tissue in the buccotemporal fascia on the medial surface of the ascending ramus. this enigmatic vestigial structure is considered to be of neuroepithelial origin with no known function. as a matter of fact, jooc is one of the most treacherous pitfalls in surgical pathology with respect to lesions in the head and neck area. hence the basic aim of this short communication is to reveal the importance about this organ and enlighten the oral pathologist about this histopathological structure, thus preventing extensive and unnecessary investigations. |
bipolar disorder (bd) is a chronic and debilitating mental disorder that affects approximately 2.4% of the general population (belmaker, 2004 ; merikangas., 2011). bipolar disorder is associated with an increased risk of mortality due to suicide (gonda., 2012) and co - morbid general medical conditions, mainly cardio - metabolic diseases (fagiolini., 2008 ; weiner., 2011) bipolar disorder is characterized by recurring major depressive, manic (or hypomanic), and mixed episodes (phillips and kupfer, 2013). lewis judd and coworkers (2002, 2003) at the national institute of mental health rated the affective symptoms of bd type i and ii patients on a weekly basis. these authors demonstrated that bd patients spend approximately half of their lifetime symptomatic, mostly with depressive episodes / symptoms. however, germane to this thesis, the course of manic depressive insanity has been noted to be characterized by significant inter - individual variation (kraepelin, 1921). in the early 1960s, leonhard collected data from 117 manic - depressive patients. predominantly manic symptoms occurred in 17.9% of patients, while 25.6% presented with a predominantly depressive clinical course, and 56.4% had equally pronounced mania and depression (leonhard, 1963). angst (1978) collected data from a sample of 95 manic - depressive inpatients from 1959 to 1975. based on this survey, angst initially proposed the concept of predominant polarity. he observed that some patients have a nuclear type of illness (i.e. patients who show both mania and depression requiring hospital admission), while some patients have predominantly depressive (i.e. the patient required hospitalization for depression but had only hypomania) or manic (the patient required hospitalizations for mania, but had no or minor depression ; angst, 1978). recently, a renewed interest in the topic of predominant polarity as a course specifier for bd emerged in the literature, driven at least in part by a corresponding observation of the differential effects of newly - discovered bipolar agents on manic and depressive features (osher., 2000 ;, 2006 ; rosa., 2008 ; colom and vieta, 2009 ; baldessarini., 2012). colom and colleagues (2006) proposed a threshold of at least two - thirds of lifetime depressive episodes for the definition of a depressive - predominant polarity, while at least two - thirds of past episodes would fulfill the criteria for mania / hypomania and define a manic - predominant polarity. however, the working definitions for predominant polarity have varied across studies (osher., 2000 ; daban., 2006 ; baldessarini., 2012). slightly more than half of bd patients exhibit a specific predominant polarity, while a significant proportion of bd patients have an undetermined predominant polarity. a previous review suggested that the depressive - predominant polarity is more common than the manic - predominant polarity (colom and vieta, 2009). however, studies which incorporated exclusively type i bd patients found the opposite pattern (i.e. a higher prevalence of the manic - predominant polarity ; osher., 2000 ; mazzarini., 2009 ; pacchiarotti., 2011 ; baldessarini., 2012). a confounder is that patterns of referral to tertiary centers where such studies are done reflect clinical acuity rather than community prevalence, providing a source of bias in such analyses. clinically - relevant correlates and outcomes for bd may be predicted by specific predominant polarity (i.e. depressive vs. manic). depressive - predominant polarity has been associated with a depressive onset of illness (rosa., 2008 ; forty., 2009 ; baldessarini., 2012), a delayed diagnosis of bd (rosa., 2008 ; baldessarini., 2012), type ii bd, and an increased risk for suicidal acts (colom., 2006 ; gonzalez - pinto., 2010 ; baldessarini., 2012). conversely, manic - predominant polarity is associated with a younger onset of illness (gonzalez - pinto., 2010 ; baldessarini., 2012), a manic / psychotic first episode (forty., 2009 ;, 2012), and a higher rate of substance abuse prior to the first episode (colom., 2006 ; popovic., 2014). furthermore, a recent systematic review identified that a depressive - predominant polarity may be associated with a higher number of mixed episodes and with the presence of melancholic features (carvalho., 2014). although a previous international society of bipolar disorders (isbd) taskforce on the nomenclature and course of bipolar disorder had concluded that the clinically - derived predominant polarity construct developed by angst (1978) and operationalized by colom. (2006) is a valid course specifier for bd, the recently - released dsm-5 did not include this concept in the criteria for bipolar disorders (apa, 2013). current views on psychiatric nosology assert that clinical utility should guide validity (mcgorry, 2013). emerging evidence indicates that the concept of predominant polarity may influence the selection of maintenance pharmacological (popovic., 2012, 2013 ; nivoli., 2013) and psychological treatments (popovic., 2013 furthermore, the polarity index metric has been recently proposed to rank maintenance treatments for bd based on the relative antidepressive versus antimanic efficacies of interventions (popovic., 2012). in keeping with this view, the overarching aim of this article was to review the extant evidence on the possible clinical utility of the concept of predominant polarity for treatment selection in bd. furthermore, we present clinical perspectives to highlight practical implications of predominant polarity. limitations of the available evidence are also discussed. a search was conducted using pubmed / medline, embase, and web of science computerized databases from inception of the project to december 8, 2013. search terms included polarity index, polarity, treatment, maintenance, predominant polarity, and bipolar disorder. original studies on the treatment implications of predominant polarity (or the polarity index) were included in this comprehensive review. popovic and coworkers (2012) previously calculated the polarity index of maintenance pharmacological treatments of bipolar disorder based on a comprehensive search of data derived from randomized controlled trials (rcts). in this report, we comprehensively searched the pubmed / medline database for new maintenance rcts of pharmacological treatments for bd from inception up to january 10, 2014. the recalculated polarity indexes of each drug (whether approved or not by regulatory agencies) are presented. the search terms bipolar disorder, mixed, mania, and bipolar depression were co - referenced with the term maintenance and the generic names of substances. eligible studies were randomized, double - blind studies which compared mood stabilizers or antipsychotic drugs, alone or in combination with standard mood - stabilizing medications (e.g. lithium and valproate), with a placebo comparator. included studies had a minimal duration of 24 weeks, in patients aged 18 years old. exclusion criteria included small sample size (i.e. a median sample size 1 would have stronger antimanic versus antidepressive prophylactic properties, while those with a pi 1 would have stronger antimanic versus antidepressive prophylactic properties, while those with a pi < 1 are more effective in preventing depressive episodes than manic relapses / recurrences. our systematic search identified 19 potentially - eligible maintenance trials for bd. a clinical trial compared the efficacy of lamotrigine + placebo versus lamotrigine + divalproex for the prevention of depressive recurrences in 86 types i and ii bd participants (bowden., 2012). however, this study did not provide data on the prevention of (hypo) manic relapses. a recent rct studied the effects of adjunctive n - acetlycysteine (nac ; 2g / day) as maintenance treatment for bd (berk., 2012). participants were initially screened for bipolar depression and had received adjunctive nac for 8 weeks (open - label phase) ; by the end of the acute phase all participants could be randomized to either nac or placebo for 24 weeks. these participants had low baseline symptoms, and the relatively low rates of recurrences possibly prevented the detection of drug differences. the authors concluded that the trial was a failed study instead of a negative one (berk., 2012). therefore, due to these limitations, this work was not included in this review. two additional rcts were identified in this updated systematic review and met inclusion criteria (woo., 2011 ; berwaerts., 2012). all investigations had an acute treatment phase, followed by a double - blind, controlled, maintenance phase. some rcts used a three - arm design : therefore, two comparisons were possible in these circumstances bd, bipolar disorder ; dss, depressive syndrome scale ; gas, global assessment scale ; hdrs, hamilton depression rating scale ; madrs, montgomery - sberg depression rating scale ; mde, major depressive episode ; rct, randomized controlled trial ; ymrs, young mania rating scale ; the nnts for the prevention of manic and depressives episodes for each agent, as well as the polarity index of each agent, are depicted in table 2. number needed to treat for the prevention of manic and depressive episodes and polarity index of drugs used for maintenance treatment of bipolar disorder. nnt values in italic are negative, indicating that placebo was more effective than active treatment, although results of original trials did not reach statistical significance. when more than one rct was available for a given treatment, calculations represent pooled results. lai, long - acting injection ; n / a, could not be calculated accurately as the nnt for the prevention of depression is negative ; nnt, number needed to treat ; rct, randomized controlled trials. a predominantly antimanic polarity index was observed for aripiprazole monotherapy (pi = 12.1 ; keck., 2007), followed by risperidone lai (quiroz., 2010 ; vieta., 2012), aripiprazole adjunctive to lithium or divalproex (marcus., 2011 ; woo., 2011), olanzapine monotherapy (tohen., 2006 ; vieta., 2012), ziprasidone adjunctive to lithium or divalproex (bowden., 2010), adjunctive risperidone lai (macfadden., 2009), and lithium (prien., 1973 ; bowden., 2000, 2003 ; calabrese., 2003 ; weisler., 2011). a polarity index favoring efficacy for the prevention of depressive episodes was observed for lamotrigine (pooled pi = 0.4 ; bowden., 2003 ; calabrese., 2003), followed by olanzapine combined with lithium or divalproex (tohen., 2004), divalproex (bowden., 2000), and oxcarbazepine combined with lithium (vieta, cruz,., 2008). adjunctive quetiapine (vieta, suppes,., 2008 ; suppes., 2009) and quetiapine monotherapy (weisler., 2011) had polarity indexes closest to unit, suggesting a more balanced efficacy for the prevention of both manic and depressive recurrences. although the primary focus of this review is on the relationships of predominant polarity (and polarity index) for the selection of maintenance drug treatments for bd, it is noteworthy that a recent systematic review also determined the polarity index for evidence - based maintenance psychological interventions for bd (popovic., 2013). in brief, most psychosocial interventions had a polarity index < 1, indicating better efficacy for the prevention of depressive episodes, including cognitive behavioral therapy (pi ranging from 0.33 to 0.89), family - focused therapy (pi = 0.42), and psychoeducation (pi ranging from 0.73 to 0.78). enhanced relapse prevention was equally effective for the prevention of depressive and manic episodes (pi = 1.0), whereas brief - technique driven interventions (pi = 3.36) and caregiver group psychoeducation (pi = 1.78) were more efficacious for the prevention of manic episodes (popovic., 2013). this review had identified three original clinical studies in which the concept of predominant polarity was investigated either as a post hoc predictor of clinical response (vieta., 2009) or as specifier associated with the selection of specific somatic treatments for bd (nivoli., 2013 ; popovic., 2014 bd, bipolar disorder ; dpp, depressive - predominant polarity ; mpp, manic - predominant polarity ; ofc, olanzapine - fluoxetine combination ; rct, ; ssris, selective serotonina reuptake inhibitors ; snris, selective norepinephrine reuptake inhibitors ; tcas, trycyclic antidepressants. a post hoc analysis of a previously published 8-week multicenter rct which compared the olanzapine - fluoxetine combination with either olanzapine alone or placebo for the treatment of bipolar i depression (tohen., 2003) investigated the relationship of predominant polarity as defined by a threshold of 2/3 of lifetime affective episodes of a given polarity (colom., 2006) on treatment response (vieta., 2009). of the 833 participants initially enrolled in the trial, 788 subjects had both baseline and follow - up ratings so as to allow final outcome assessments ; of these patients, 367/788 (46.6%) could be categorized as having had either a depressive - predominant polarity (269/788 ; 34.1%) or a manic - predominant polarity (98/788 ; 12.4%). the primary measure was a change in the clinical global impression of severity of major depression (cgi - d ; spearing., 1997). the effect of predominant polarity as an outcome predictor was markedly dissimilar between men and women. in women there were no significant differences in cgi - d scores in accordance with predominant polarity (i.e. manic versus depressive), whereas in men the predominantly manic group has a significantly better outcome when compared to the predominantly depressive group. furthermore, predominantly manic men had better outcomes compared to women with both predominant polarity types (vieta., 2009). the prescription patterns of a sample of 604 dsm - iv - tr bd patients attending the bipolar unit of barcelona were investigated in a naturalistic study. a principal component analysis showed three factors : (i) an antimanic stabilization package, which was characterized by the use of classic thymoleptic medications (i.e. lithium, valproate, and carbamazepine), three atypical antipsychotics (clozapine, risperidone, and olanzapine), and electroconvulsive therapy ; (ii) an antidepressive stabilization package, which included lamotrigine and other atypical antipsychotic agents (notably quetiapine) ; and (iii) an anti - bipolar ii package, which included diverse antidepressants. bipolar patients with a predominantly manic polarity were treated mainly with the antimanic stabilization package, whereas bd patients with a depressive - predominant polarity were more frequently treated with the antidepressive stabilization package (nivoli., 2013). the anti - bipolar ii package included mainly type ii bd patients with a depressive - predominant polarity (nivoli., 2013). popovic and colleagues (2014) studied a sample of 604 dsm - iv - tr bd patients, of which 257 presented a clear predominant polarity type (n = 143 ; 55.6% had a depressive - predominant polarity and n = 114 ; 44.4% had a manic - predominant polarity ; colom., the total polarity index (calculated as mean value of polarity index of all prescribed mood stabilizers and antipsychotics in each patient) were significantly higher in the predominantly manic group. moreover, the polarity index of antipsychotics and mood stabilizers taken separately were also higher in the manic - predominant polarity group. the use of antidepressants, lamotrigine, and benzodiazepines was more prevalent in the depressive - predominant polarity group (popovic., 2014). brugue and vieta (2007) had previously hypothesized that a lower d2 receptor occupancy would be more more relevant than a greater 5-ht(2) receptor action to the efficacy of atypical antipsychotics for the acute treatment of bipolar depression herein we examined the relationship of d2 receptor affinities (richtand., 2007) and the polarity index of atypical antipsychotrics in monotherapy (table 4). overall, we found that the higher the d2 receptor binding affinity, the lower the pi of different atypical antipsychotics in monotherapy. aripiprazole did not seem to follow the same pattern : in fact, the mechanism of action of aripiprazole is rather complex (mailman and murthy, 2010). once thought to primarily act as a partial agonist at d2 receptors, new data support that aripiprazole may act either as an antagonist or as an agonist at distinct subpopulations of d2 receptors in a process referred to as functional selectivity (mailman and murthy, 2010). these unique mechanisms may explain the divergent findings related to d2 binding affinity and the pi of this compound. polarity index based on monotherapy of atypical antipsychotics and dopamine d2 receptors. ki values obtained from richtand. ki, dissociation constant ; lai, long - acting injection ; nnt, number needed to treat. in spite of the fact that the efficacy of antidepressant drugs for the acute and maintenance treatment of bd is one of the most long - standing controversial topics in the literature (gitlin, 2012 ; malhi, 2012), in part due to an inconsistent evidence base (vazquez., 2011), epidemiological surveys indicate that antidepressant drugs are widely employed in the management of bd, mainly for the management of acute bipolar depression episodes (post., 2011 ; this status quo resulted in a recent isbd consensus panel on the use of antidepressant in bd (pacchiarotti., 2013), based to a large degree on expert consensus through the delphi method. however, the evidence reviewed thus far suggests that clinicians from tertiary mood disorder services consider the patient s predominant polarity while prescribing antidepressants. given the extant evidence base showing that maintenance agents differ in their efficacy across the two poles, the predominant polarity of a given patient is probably considered by clinicians more broadly when selecting both acute and long - term maintenance treatments. as stated before, there are concerns regarding the use of antidepressants for the long - term treatment of bd. notwithstanding concerns about the so - called antidepressant - induced manic switch, this aspect remains controversial, as a clear causal relationship linking antidepressant exposure to the precipitation of mania is not firmly established (licht., predominant polarity predicts several clinical correlates in bd, as reviewed elsewhere (colom and vieta, 2009 ; baldessarini., 2012). the present review strongly suggests that the concept of predominant polarity is useful for the selection of appropriate maintenance treatments for bd. in keeping with this view, it is important to consider that one should consider the clinical utility of a given specifier to endorse its inclusion in current psychiatric nosology (mcgorry, 2013). this review demonstrates that different maintenance drug treatments for bd may have differential efficacies for the prevention of manic versus depressive episodes. this systematic literature search updates a previous review (popovic., 2012). we had identified two additional eligible rcts (woo., 2011 ; berwaerts., 2012). importantly, paliperidone s pi could not be calculated, as the nnt for the prevention of depressive episodes was negative. hence, most atypical antipsychotics have a greater efficacy for the prevention of manic episodes, while quetiapine has a more balanced efficacy for the maintenance treatment of bd. the published post hoc analysis of the 8-week rct, which studied the olanzapine - fluoxetine combination versus both olanzapine alone and placebo in the acute treatment of bipolar depression (vieta., 2009), found that a manic - predominant polarity predicted efficacy specifically for male participants. while these data suggest that predominant polarity may influence acute treatment responses in bd, the limitations of this study do not allow the establishment of firm directions. in fact, most patients in that trial had a 2.7-fold excess for the depressive - predominant polarity when compared to a manic - predominant polarity, as expected from a recruited sample for a bipolar depression trial. furthermore, this post hoc analysis could not include all participants of the original trial (tohen., 2003). thus, one might argue that this analysis did not have adequate power to detect additional inferences, as acknowledged by the authors (vieta., 2009). consequently, the role of predominant polarity for the prediction of acute treatment responses in bd remains, to a large extent, an open question. two naturalistic clinical studies from the same research group (nivoli., 2013 ; popovic., 2014) suggest that the concept of predominant polarity may play a decisive role for the selection of maintenance pharmacological treatments for bd. the first report showed that patients with a predominantly manic polarity would be treated in the majority circumstances with an antimanic stabilization package, while patients with a depressive - predominant polarity would be mainly treated with an antidepressive stabilization package (nivoli., 2013). patients with a manic - predominant polarity were treated with a therapeutic regimen with a higher mean polarity index, while patients with a depressive - predominant polarity were treated with agents with an overall lower pi (popovic., 2014). despite the fact that these data may provide some support for the wise everyday care of bd first, the data came from a single center (i.e. the barcelona bd center). therefore, it should be replicated in prospective multi - center studies to establish more conclusive evidences on the true role of predominant polarity (and the pi) for the selection of maintenance treatments for bd. lastly, there is a degree of circularity in the argument : clinicians choose treatments they believe to be most useful for an individual s pattern of illness, and it is unsurprising that prescription patterns reflect this. although intuitive and easy - to - use, the pi has been criticized as imperfect by some investigators (alphs., 2013). second, there are variations in clinical and socio - demographic characteristics of included participants across different trials (for example, age, sex, previous exposure to treatment, comorbidities, number of previous affective episodes, etc). third, the calculation of the pi would be impossible when confidence intervals of nnts for the prevention of either depressive or manic episodes includes zero. lastly, it is relativistic, not absolute ; an agent that has both potent anti - manic and anti - depressive effects would have the same pi as an agent weak in both poles. we agree with the authors that no simple metric is sufficient for the complex evidence - based final choice of a given maintenance treatment for bd. for instance, consider that a drug with a nnt for prevention of mania of 4.0 and a nnt for the prevention of depression of 2.0 would have a pi = 2.0, whereas a drug with a nnt for the prevention of mania of 8.0 and a nnt for the prevention of depression of 2.5 would have the same pi = 3.2. this would induce the clinician to prescribe the latter drug for use in the maintenance treatment of a predominantly manic patient. we argue here that the pi provides useful, albeit imperfect, information for the choice of a maintenance treatment of bd for a given patient. for example, the pi should be used while considering the nnt and nnh (i.e. number needed to harm), and also taking into account the methodological quality (and risk of bias) of isolated rcts that contributed to the pi, nnt, and nnh estimations. furthermore, the calculated pi for divalproex and oxcarbazepine should be interpreted with extreme caution because pivotal trials did not evidence superiority over placebo (bowden. an important limitation of the current concept of predominant polarity (and the polarity index) refers to the fact that mixed episodes are not contemplated. mixed episodes have been increasingly emphasized in the current nosology of bd (berk. therefore, future studies should investigate the possible existence of a predominantly mixed type of bd. this awaited evolution in the concept of predominant polarity may provide a substrate to add potentially valuable pieces of information to the selection of the limited alternatives available for the treatment of mixed affective episodes (fountoulakis., 2012). a first approach to this concept has been conducted (pacchiarotti., 2011). the present review concludes that the concepts of predominant polarity and polarity indexes provide clinically useful information for the selection of maintenance treatments for bd. furthermore, there are important unanswered questions for further research and this opens an important debate for the incorporation of these constructs in current psychiatric nosology. if one considers clinical utility as a determinant for the construction of nosology, this work concludes that there probably is sufficient evidence for the incorporation of predominant polarity as a course specifier for bd. however, clinical trials (both acute and maintenance) may include the baseline determination of predominant polarity as a potential predictor of response. pooled analyses would ultimately provide a stronger evidence base. however, there are no published studies that we are aware of investigating biological leads (e.g. genetic polymorphisms, neuropsychological tests, and neuroimaging correlates) related to predominant polarity. considering emerging neuroscience - based models of psychiatric nosology (such as the national institute of mental health research domain criteria), further investigations are needed in this particular field (insel., 2010 ; cuthbert and insel, 2013). dr mcintyre has received research grants from stanley medical research institute, national alliance for research on schizophrenia and depression (narsad), national institutes of mentalhealth, eli lilly, janssen - ortho, shire, astra - zeneca, pfizer, lundbeck, forest, sepracor, and bms ; has served on advisory boards for astra - zeneca, bristol - myers squibb, janssen - ortho, eli lilly, lundbeck, pfizer, shire, merck, and sepracorand otsuka ; has served on speaker bureaus for janssen - ortho, astra zeneca, eli lilly, lundbeck, merck, pfizer, and otsuka ; and cme activities for astra zeneca, bristol - myers squibb, physicians postgraduate press, cme outfitters, merck, eli lilly, pfizer, lundbeck and otsuka. dr fountoulakis is or was a member of consultation boards for several pharmaceutical companies and has received honoraria for lectures and support for participating in congresses from astrazeneca, bms, janssen - cilag, and eli - lilly. dr berk served as a consultant to astra zeneca, bristol myers squibb, eli lilly, glaxosmithkline, janssen cilag, lundbeck, and servier ; is on the speaker s bureaus of astra zeneca, bristol eli lilly, glaxosmithkline, janssen cilag, lundbeck, pfizer, sanofi, synthlabo, servier, solvay, and wyeth ; has received grant and/or research support from astra zeneca, beyond blue, bristol myers squibb, eli lilly, glaxosmithkline, janssen cilag, lundbeck, mayne pharma, mbf bioscience, national health and medical research council, novartis, organon, servier, and stanleymedical research foundation ; and has received honoraria from astra zeneca, bristol myers squibb, eli lilly, glaxosmithkline, janssen cilag, lundbeck, pfizer, sanofi, synthlabo, servier, solvay, and wyeth. dr vieta has received grants, acted as a consultant, or served on advisory boards for alexza, almirall, astrazeneca, bristol - myers squibb, eli lilly, ferrer, forest, glaxosmithkline, janssen, jazz, johnson & johnson, lundbeck, merck - sharpe & dohme, novartis, organon, otsuka, pfizer, richter, sanofi, servier, schering plough, shire, takeda, teva, united biosource corporation, and wyeth. | background : bipolar disorder (bd) is a serious and recurring condition that affects approximately 2.4% of the global population. about half of bd sufferers have an illness course characterized by either a manic or a depressive predominance. this predominant polarity in bd may be differentially associated with several clinical correlates. the concept of a polarity index (pi) has been recently proposed as an index of the antimanic versus antidepressive efficacy of various maintenance treatments for bd. notwithstanding its potential clinical utility, predominant polarity was not included in the dsm-5 as a bd course specifier.methods:here we searched computerized databases for original clinical studies on the role of predominant polarity for selection of and response to pharmacological treatments for bd. furthermore, we systematically searched the pubmed database for maintenance randomized controlled trials (rcts) for bd to determine the pi of the various pharmacological agents for bd.results:we found support from naturalistic studies that bipolar patients with a predominantly depressive polarity are more likely to be treated with an antidepressive stabilization package, while bd patients with a manic - predominant polarity are more frequently treated with an antimanic stabilization package. furthermore, predominantly manic bd patients received therapeutic regimens with a higher mean pi. the calculated pi varied from 0.4 (for lamotrigine) to 12.1 (for aripiprazole).conclusions : this review supports the clinical relevance of predominant polarity as a course specifier for bd. future studies should investigate the role of baseline, predominant polarity as an outcome predictor of bd maintenance rcts. |
according to the definition of the world health organization (who) health is not just the absence of disability or disease but a full sense of physical, mental and social wellbeing (1). according to the who, 70 - 80% of deaths in developed countries and 40 - 50% of deaths in less - developed countries are caused by diseases emerging due to lifestyle. the way to prevent disease related to lifestyle, such as heart disease, obesity and diabetes, is to change lifestyle behavior (2). healthy lifestyle behavior (hlb) comprises the methods used by individuals to stay healthy and prevent disease along with their beliefs about health. according to another paper, healthy lifestyle comprises all approaches used by an individual to take control of behavior related to themselves and to raise the health levels of daily activities. healthy lifestyle behavior then is behavior motivated by protecting the state of well - being and keeping well - being at high levels (4). according to the center for disease control (cdc), gaining and applying these are insufficient physical activity, bad nutrition, tobacco and use of tobacco products and alcohol intake (5). these unhealthy behaviors are responsible for many diseases and the development of deadly chronic diseases. according to data from the turkish statistical institute (tsi), circulation system diseases (39.8%), benign and malignant tumors (21.3%), respiratory diseases (9.8%) and diseases related to endocrine, nutrition and metabolism (5.6%) take first place among diseases causing death. of circulation system disease deaths 38.8% are due to ischemic heart disease, 25.2% are due to cerebrovascular diseases, 17.7% are due to other heart diseases and 12.8% are due to hypertensive diseases (6). the leading risk factors playing a role in the development of cardiovascular diseases are cigarettes, inactive lifestyle and unhealthy nutrition. of every 10 americans who die each year, the cause of death in 7 is chronic disease. heart disease, cancer and stroke are responsible for more than 50% of deaths each year (7). as a result to reduce these preventable diseases in society, as indicated, there is a strong relationship between chronic diseases and the lifestyle of individuals (8). currently it is recommended to identify risk factors related to lifestyle, direct individuals to develop healthy lifestyle behavior and correct negative environmental conditions (8 ; 9 ; 10 ; 11). on this point,, it is very important to repeat health education for each age group at regular intervals. among these age groups, young adults take first place. to prevent chronic diseases identifying risk behavior that plays a role in developing unhealthy lifestyles and to take early precautions with education, it is easier, albeit relatively, to change unhealthy behavior in the university period than in later life. university life is a period when important changes occur and risk behavior can develop. apart from career education in this period, there are changes in physical and personal development, in the psycho - social life and health behavior of the individual. university students are an important risk group expected to practice healthy lifestyle behavior to protect and develop societal health. developing healthy attitudes and behavior in students will contribute to the development of healthy lifestyle behavior in society (8;12). this study aimed to investigate the incidence and risk factors of healthy lifestyle behavior in a female dormitory in krehir. this descriptive study was completed in a female dormitory between march and may 2014 in a state in the central anatolia region. before choosing the sample the population was studied, however some students did not want to answer the survey forms and as some forms were not fully completed, 295 students were included in the study. permission for the research was obtained from the necessary institutions and, with respect for voluntary principles, written consent was obtained from the students participating in the study. data was collected with a personal information form and the healthy lifestyle behavior scale. the personal information form comprised 20 questions aiming to determine the sociodemographic characteristics of the students and their perception of healthy lifestyle. the healthy lifestyle behavior scale (hlbs) was developed by walker. in 1987. the validity and reliability study for our country was completed by esin in 1997 (13). the subscales are personal development, health responsibility, exercise, nutrition, interpersonal support and stress management. never 1 point, sometimes 2 points, often 3 points and regularly 4 points. the lowest points for the whole scale are 48 while the highest points are 192. as the points increase the positive health behavior levels increase. in the present study, dependent variable was hlbs score and its subgroups. while independent variables were age, class grade, section, family type, place of living, parents education and occupation, income perception, sports, academic achievement, body mass index, nutrition type. the variables were investigated using visual (histograms, probability plots) and analytical methods (kolmogorov - simirnov / shapiro - wilk s test) to determine whether or not they are normally distributed. descriptive statistics (frequencies, percentages, means, standard deviations) were used to describe groups of numerical data and the basic features of the data. independent samples t test and kruskal wallis test were used to compare numerical parameters beteen the groups. as seen in table 1, 34.2% of participants (n:101) were studying in the nursing department and 47.5% (n:140) were first year students. the place of longest residence was city for 29.8% (n:87) and metropolis for 29.5% (n:88). the perception of health was described as good by 53.2% of students (n:157), 7.5% (n:22) used cigarettes, 2.4% (n:7) consumed alcohol, and 87.1% (n:257) ate a mainly mixed diet. according to bmi, 80.3% (n:237) were normal and 12.9% (n:38) were underweight. to pass the time 30.8% (n:91) of students used computers and the internet, 29.2% (n:86) used books and music and it was found that 81.0% (n:239) of students participating in the study lived in a nuclear family setting. the mothers of 64.4% (n:190) and the fathers of 59.0% (n:174) were primary school graduates with the mothers of 88.1% (n:260) housewives, the fathers of 24.7% (n:73) working in the private sector and the income of 69.5% (n:205) at middle levels. the subscales and total points averages from the healthy lifestyle behavior of students participating in the study are presented in table 2. healthy lifestyle behaviour scale scores of the study population, krsehir, 2014. as seen in table 3, when the healthy lifestyle behavior of students in the study is investigated according to perception of health, alcohol intake and bmi, there is no statistically significant difference found (p>0.05). the exercise and total healthy lifestyle behavior of those who do regular sport and the average nutrition points of those who do not consume cigarettes were found to be higher (p0.05). when the department attended is investigated, the average health responsibility, exercise and healthy lifestyle behavior averages are found to be higher in undergraduate students compared to certificate students and this difference was found to be statistically significant (p0.05). distribution and comparison of healthy lifestyle behaviour scale scores obtained by department, class, academic achievement and civic location. this descriptive study completed in a female dormitory in krehir investigated the healthy lifestyle behavior (hlbs) of students. the positive health behavior of individuals is related to gaining a high score on the hlbs and subscales. in our study the average hlbs points of the students were 120.2416.99. in a study by imek. the average points were 134.4 9.7 ; while studies in the states of orum, anakkale and hatay had similar average points to our study (121.5719.65 ; 122.119.8 and 122.09 16.93, respectively) (14 ; 15 ; 4). the average points for university students in turkey from other studies range from 116.1 to 126.4 (16, 17). the majority of students in our study group came from nuclear families and this sociodemographic finding was similar to the anakkale study (15). the majority of students in our working group were studying in the nursing department, together with female students of the vocational health school. around half of participants had good academic success, the body mass index of the majority was normal and cigarettes use was very low. in this study a statistical difference was found between the dependent variables of healthy lifestyle behavior and subscales and the independent variables of regular sport, cigarette use, and academic success levels (p0.05). those living with family, those from broken homes, those with health perceived as middling and those with perceived high level economic situation had better healthy lifestyle behavior (16). additionally when the average points for subscales of the healthy lifestyle behavior scale are examined, nurses were found to have highest points for self - realization and the lowest points for exercise, nutrition and stress management subscales, in that order (18). also in a study of university students, health responsibility and nutrition average points of the healthy lifestyle behavior scale for nursing students were found to be higher at a statistically significant level compared to students from other departments. however the same study found that students in the business, nursing and science - literature departments had lower average points for the exercise subscale compared to students from other departments (2). according to these results while nutritional habits are better in the student years, after entering working life due to the effect of work stress, factors related to working conditions or stress factors from family - social environments, nutritional habits may got worse with age and the risk of developing unhealthy nutritional behavior should not be forgotten. another important healthy lifestyle behavior of regular physical activity was found to be low among nursing students or in studies of nurses, creating another important work health problem (18 ; 2 ; 4). regular education directed at developing and maintaining exercise behavior, an important protective factor against obesity and the struggle with work stress among health workers, should be provided. a study by koolu and akn in konya city center investigated the effect of socioeconomic inequality in health and healthy lifestyle behavior and quality of life. in conclusion they showed that class position, perceived income level, place of residence and number living in the household had a determining role in healthy lifestyle behavior and quality of life (27). ilhan. in a study of university students found that the health responsibility and nutrition subscale average points were significantly higher for students in the nursing department compared to students from other departments (2). the same study found the hlbs average points were higher to a statistically significant degree for those with very good economic situation compared to those with middling and good economic situation. in our study those with bad economic situation had exercise and interpersonal support point averages statistically significantly higher than those with middling or good economic situation. an educational study of students in a vocational health school determined a significant increase in the level of healthy lifestyle behavior from before education to after education (15). additionally when the cost of time and effort to create behavior changes is considered, education to develop positive health behavior during the student period will be very productive for risk groups such as nurses who will provide service as health professionals. gner and demir in a study of surgery nurses found average healthy lifestyle behavior points of 116.8916.3. they also emphasized that nurses, whose education is related to health, should have higher points (28). we consider that regular and continuous education to develop healthy lifestyle behavior during undergraduate education will benefit students who will become the health professionals of the future. additionally monitoring to determine the sociodemographic needs of students who may have disadvantages such as in our study group and inclusion in education to develop health will be helpful to develop positive health behavior. | goal : this study is a descriptive analysis aiming to determine the healthy lifestyle behaviors of students staying in a female dormitory in the central anatolia region.methods:a total of 295 students staying in a state - run female dormitory were included in the study. data was collected with a personal information form and a healthy lifestyle behavior scale between march and may 2014. the dependent variables of the study were the hlbs points and subscales. the independent variables were age, class, department, family structure, place of longest residence, family education and occupation, perception of income, sport, academic success, bmi and nutrition.results:the average age of students participating in the study was 19.921.39 (17 - 26) years. the average hlbs points of the students in the study were determined to be 120.2416.99 (85 - 170). there was a statistically significant difference found between the students regular participation in sport, use of cigarettes, department they studied in and academic success levels with healthy lifestyle behavior and subscales (p<0.05).conclusion : we determined that student scores taken from healthy lifestyle behaviors scale was moderate level. we consider that regular and continuous education to develop healthy lifestyle behavior during undergraduate education will benefit students who will become the health professionals of the future. |
total knee arthroplasty (tka) is considered to be a successful orthopedic procedure for pain relief and improvement of the physical function of patients with severe knee osteoarthritis1. however, the edematous response to traumatic orthopedic procedures such as tka creates incompetence in the lymphatic system and persistent edema2. therefore, tka is followed by a convalescence period, during which undesirable sequelae such as edema and postoperative pain may limit early functional recovery. the effect of swelling, inflammation, and pain on muscle inhibition has been well documented3, 4. endermologie delivers mechanical massage to the limb via two motorised, cylindrical skin rollers which pick up and massage the skin inside its treatment head. previous studies of this equipment have shown that it improves superficial lymphatic drainage and lymphatic transport capacity5. to our knowledge, the benefits of endermologie as an edema treatment method following tka have not been evaluated. the aims of this study were to investigate the efficacy of endermologie, in the early postoperative period after tka, in reducing edema and knee pain, and improving active knee range of motion in comparison with conventional postoperative care using various objective outcome measurements. eighteen patients with end - stage knee osteoarthritis (5 males and 13 females ; mean age, 73.4 5.7 years) scheduled for primary unilateral tka between august 2014 and may 2015 were selected for this study. patients were excluded if they had an active infection, major cardiac pathology, or thrombus or venous obstruction that was prediagnosed or revealed on a routine preadmission hospital screening6. this study was approved by the ethics committee of the jeju national university of korea and written consent to participication in the study was obtained from all of the subjects in accordance with the declaration of helsinki. eight patients were allocated to the intervention group, and ten patients to the control group. all patients underwent tka performed by a single experienced orthopedic surgeon specializing in joint replacement surgery. a midline incision and the medial parapatellar retinacular approach were used in all cases. on the seventh day postsurgery, patients allocated to the intervention group underwent a 20 minutes of endermologie therapy on the operated limb applied by a physical therapist trained in the technique ; the cellu - m50 lpg systems device (lpg systems, valence, france) was used. the procedure consists of a tissue mobilization process between two rollers, creating a skin fold and stretching the underlying tissue to improve superficial lymphatic drainage. focus was initially on the proximal tissue, gradually moving distally to the area around the knee, popliteal region, and back proximally, to optimize the lymphatic system by clearing lymphatic drainage in areas adjacent to the regions of knee edema, and develop new pathways for travel2, 7. this process of working proximal to distal and then distal to proximal was repeated 3 to 4 times over the 20-minute treatment period. participants in the control group received conventional physiotherapy including cryotherapy and pneumatic leg pump therapy for the same amount of time as the intervention group. both groups received their treatments 5 days a week (5 treatment sessions in total). postoperative inpatient rehabilitation such as isometric contraction of the quadriceps and gluteal musculature was standard for all of the study patients. the following clinical measures were evaluated before and after one week of treatment by the patient s allocated physician who was blinded to the treatments. first, active knee flexion and extension were measured using a handheld goniometer, creating an angle made by three anatomical landmarks : the greater trochanter of the femur at the hip, the lateral femoral condyle at the knee, and the lateral malleolus at the ankle7. second, knee pain severity was evaluated using a numeric rating scale (nrs), on a whole number rating scale from 0 (no pain) to 10 (worst pain). third, the circumferences of the operated limbs at 10 cm above and below the midpatella were assessed (suprapatellar circumference and infrapatellar circumference). measurements were taken twice with a tape measure at each site, and the minimum value was recorded8. fourth, ultrasonography was performed on the operated limb of each participant using a 9.0 mhz transducer (ge healthcare, milwaukee, wisconsin). the superior, medial, inferior and lateral directions were marked to measure the desired cross section at 10 cm above the midpatella. the soft tissue cross - sectional area was calculated as described by hwang. finally, the ratio of extracellular fluid (ecf) volume, and the ratio of the single frequency bioimpedance analysis (sfbia) value at 5 khz of the bilateral lower extremities was evaluated using bioelectrical impedance spectroscopy (inbody 720 biospace, seoul, south korea)10. the ecf ratios of the operated to the non - operated side and the non - operated to the operated side ratio of the sfbia value at 5 khz were calculated. a lower value of sfbia at 5 khz suggests that there is fluid in the extracellular space, and higher ratios of both calculated ecf and sfbia suggest that there is more ecf in the operated limb10. the change in each outcome measure from pre- to post - treatment the change in each outcome measure from pre- to post- treatment was compared between the two groups using the mann - whitney test. statistical analyses were performed using spss for windows version 20 (ibm - spss, inc., patient demographics were similar between the two groups and are summarized in table 1table 1.baseline demographic characteristics of the participantsvariablesintervention group control grouplesion side, right / left 4/44/6gender, males / females3/5 2/8age (years)70.8 5.475.6 5.1height (cm)155.9 8.2155.2 7.9weight (kg)64.6 6.966.1 10.0body mass index (kg / m)26.7 3.2 27.3 2.8values are numbers or mean standard deviation. the intervention group showed significant improvement from pre- to post - treatment in active knee flexion, knee pain, both circumferences of the operated limb and cross - sectional area of soft tissue at 10 cm above the midpatella, and the ratio of the sfbia value at 5 khz (table 2table 2.changes in knee range of motion, pain and edema of the intervention and control groups before and after treatmentvariablesintervention groupcontrol groupintergroup difference beforeafter intragroup differencebeforeafterintragroup differencee - arom () 13.1 7.011.9 6.50.1614.08.113.06.70.080.82f - arom () 73.119.388.818.70.02 74.011.391.010.80.01 0.79nrs3.50.52.10.80.02 3.70.92.60.50.01 0.34sc (cm)42.33.641.4 3.30.03 43.73.341.82.80.01 0.18ic (cm)33.82.032.92.20.0634.92.634.22.30.04 0.74csa (mm)49.813.139.913.20.01 50.79.143.78.90.01 0.31recf (%) 104.32.9103.31.80.23104.42.4101.46.90.180.65rsfbia (%) 134.816.0123.66.90.04 136.417.3126.114.10.01 e - arom : extension active range of motion ; f - arom : flexion active range of motion ; nrs : numeric rating scale ; sc : suprapatellar circumference ; ic : infrapatellar circumference ; csa : cross - sectional area of soft tissue ; recf : the ratio of extracelluar fluid volume ; rsfbia : the ratio of single frequency bioimpedance analysis. no statistically significant between - group differences were found for any of the variables before and after treatment ; however, the infrapatellar circumference of the surgical limb showed a significant reduction only in the control group (table 2). although not statistically significant, a decreasing trend was exhibited for the infrapatellar circumference in the intervention group. e - arom : extension active range of motion ; f - arom : flexion active range of motion ; nrs : numeric rating scale ; sc : suprapatellar circumference ; ic : infrapatellar circumference ; csa : cross - sectional area of soft tissue ; recf : the ratio of extracelluar fluid volume ; rsfbia : the ratio of single frequency bioimpedance analysis. p<0.05 this is the first study to investigate the therapeutic effect of mechanical massage for patients with knee edema after tka in the early postoperative period, and to compare the effectiveness of conventional physical therapy and mechanical massage using objective measures of soft tissue edema change. the findings of the present study are in line with the findings of ebert., who also studied the efficacy of massage intended to encourage the natural drainage of the lymph in early outcomes after tka, in addition to conventional care7. however, the present study is the first to show the effect of mechanical massage via endermologie alone in the absence of other physical therapies. in addition, the objective measurements of the present study included the amount of soft tissue in the cross - sectional area around the knee as assessed by using ultrasonography, and the ratio of extracelluar fluid volume and the ratio of sfbia value at 5 khz, as well as limb circumference, and is, therefore, methodologically different from the study of ebert. massage is known to manipulate the attachments below the skin, eliciting modification in the connective tissues11. endermologie is an original noninvasive technique, consisting of a delicate and reproducible mechanical massage, and elicits a profound physiological alteration in lymphatic flow5, 12. a previous study demonstrated the effect of mechanical massage via endermologie on the treatment of limb - swelling in patients with secondary arm lymphoedema5. in line with the methods of this previous study, we included endermologie to treat limb edema. however, there is a difference in the populations of the two trials, as the subjects in the study of moseley., had secondary arm lymphoedema5, whereas subjects in the present study had knee edema following tka. knee edema is a normal body response to surgical trauma and occurs in most patients who undergo tka13, 14. the consequences of knee edema are pain, inflammation, decreased range of motion, quadriceps inhibition, functional limitation and a delay in rehabilitation13, 15. in addition, fear of symptom aggravation typically precludes progressive strength training early after tka16. despite the influence of knee edema on the rehabilitation process there is evidence to support the effect of early and intensive exercise after tka in the prevention of early loss of muscle strength and function after surgery3, 17. therefore, adopting mechanical massage treatment in the early postoperative period could potentially benefit patients rehabilitation. it is the first study to provide objective evidence of edema reduction by mechanical massage using ultrasonography and bioelectrical impedance spectroscopy. muscular atrophy is substantial following tka ; however, limb circumference measurements do not allow for the differentiation between respective variations in edema and muscular mass18. in contrast, ultrasonography and bioelectrical impedance spectroscopy can measure limb edema independently, and have excellent reliability and accuracy9, 13. also, endermologie was used as a specific type of mechanical massage in comparison with other conventional physical therapies. in addition, our findings open a new possibility in the early management of knee edema after tka to improve the early rehabilitation process. a significant improvement in active knee flexion was observed at the follow - up assessment time prior to hospital discharge, after one week of endermologie treatment sessions, in the present study. findings from previous studies highlighted the importance of early attainment of active knee flexion for long - term actual and perceived benefits7. first, this was a pilot study including a small number of patients that aimed to evaluate the effect of mechanical massage on knee edema early after tka. second, there was no follow - up ; therefore, the long - term effects of mechanical massage on edema, pain and range of motion after discharge remain unclear. in conclusion, the results of this pilot study indicate that mechanical massage via endermologie is effective in knee edema treatment following tka, leading to better early outcomes for the patients. | [purpose ] the aim of this study was to evaluate the efficacy of mechanical massage via endermologie after total knee arthroplasty in reducing edema and pain and improving knee range of motion, in the early postoperative period. [subjects and methods ] eighteen patients with knee edema following total knee arthroplasty were randomly assigned to the intervention group (n=8) or the control group (n=10). the intervention group received mechanical massage therapy using endermologie and the control group received conventional physical therapy for 20 minutes a day, 5 times a week from the seventh day postsurgery. clinical assessments included active knee flexion and extension range of motion, knee pain using a numeric rating scale, the operated limb circumference, the soft tissue cross - sectional area using ultrasonography, the extracelluar fluid volume, and single frequency bioimpedance analysis at 5 khz using bioelectrical impedance spectroscopy. [results ] both groups showed significant reduction in edema and pain, and improvement in active knee flexion at the end of treatment. there were no significant inter - group differences before or after treatment. [conclusion ] mechanical massage could be an alternative way of managing knee edema after total knee arthroplasty in early postoperative recovery. |
suicide by self - stabbing is uncommon, constituting only 1 - 3% of suicide attempts. patients with self - inflicted stab wounds may have a higher incidence of surgical interventions based on the stab location. there are only a few case reports and small series published in the literature of the subject. a 48-year - old female patient was admitted to the emergency department due to a stab wound of the neck region (fig. the rescue team leader informed us that the patient had tried to commit suicide. position of retained blade after the completion of the initial medical history, a thorough physical examination was conducted to detect any major vital organ damage to the neck and concomitant injuries. there was no presence of active bleeding, no bruit, no pulsatile hematoma, no sign of central neurological deficit, hemoptysis, stridor, hoarseness, dysphagia or subcutaneous emphysema. the patient, with full cardiopulmonary viability, was transported to the radiology department to conduct a complete computed tomography (ct) scan of the neck and thorax. we were quite astonished with the results, which showed that the knife reached completely through the central mediastinum to the posterior mediastinum, attaining the aortic arch, gently leaning on the vessel 's adventitia, and not damaging in the process any vital structure (fig. 2 a and b). the ct scan revealed a bilateral pneumothorax, due to which bilateral drainage of the pleural cavity was administered through the second intercostal space in the midclavicular line using thorax 24 f drains (fig. position of retained blade in 3d ct bilateral pneumothorax a left - sided anterolateral thoracotomy was conducted in the supine position. the access limited the direct view into the posterior mediastinum, but our idea was to reduce any patient rotation to a minimum, so that the injury to the vascular wall of the aorta was not compounded. after ensuring that the tip of the blade did not penetrate completely through the aortic vessel wall, but only bordered the surface of adventitia of the aortic arch, the first assist removed the knife. subsequently to the removal of the blade, no hemorrhage to the pleural cavity was noted, and at this stage the decision to leave an additional drain in the left pleural cavity and to close the thorax was made. afterwards the neck injury exploration was begun. to extend the post - injury wound a cut along the medial edge of the right sternocleidomastoid muscle was applied. during the dissection towards the trachea no damage to the vital anatomic structures was noted. however, after the deep layers behind the trachea were explored, tangential damage to the tunica adventitia of the esophagus was discovered. a redon drain was left in the attended injury site, which was removed on the second day after the surgery. the upper drains from the pleural cavity were removed on the third day after surgery, and the left lower drain on the second day. the patient was discharged to a psychiatric hospital on the seventh day after the surgery. during the follow - up visit on the tenth day penetrating neck trauma is responsible for less than 5% of all trauma admissions. these injuries are unique and challenging, especially those which penetrate to the thorax cavity. the abbreviated surgical exploration is also in many cases insufficient to answer whether an extended surgical procedure is necessary. according to roon and christensen, the neck can be divided into three zones : zone i between the sternal notch and the cricoid cartilage ; zone ii between the cricoid cartilage and the lower border of the mandible ; and zone iii between the lower border of the mandible and the base of the skull. up to 80% of injuries involve zone ii. according to international literature, the most frequent site of injury is the aerodigestive tract, followed by the major vascular structures and nerve injuries [2, 3 ]. although zones i and iii are protected by bones, penetrating trauma to this area is more dangerous than to zone ii because of the proximity of the thorax and skull base. when lack of symptoms and absence of neck swelling do not exclude the possibility of underlying serious injuries, the knowledge of the common consequences of each zone of injury can provide some idea of the possible extent of the internal damage. hemodynamically stable patients require extended diagnostic procedures to plan the final surgical procedure. in this particular case, where our patient showed full cardiopulmonary viability, extended radiological diagnostics (the ct scan of the neck and thorax) allowed detailed visualization of the trajectory of the blade, which was observed to be 14 cm in length and penetrated into the posterior mediastinum, bordering the vessel wall of the aorta. this diagnostic method also made the concomitant injury of the bilateral pneumothorax apparent, although it did not reveal the damage to the tunica adventitia of the esophagus in the cervical region. this case is extremely unusual for at least two reasons : firstly, the self - inflicted stab wound is an uncommon method of suicide attempt, and secondly, to date we have not come across an analysis of a wound penetrating from the neck region through the central mediastinum to the posterior mediastinum, reaching the vessel wall of the aortic arch and not damaging any vital structures in accessible databases of international literature. medical management in such cases, due to their infrequency, requires, besides the individual approach, excessive caution and control. no unified recommendations for such injuries have been established yet [4, 5 ]. the importance of using a spiral ct scan in hemodynamically stable patients is underlined in the international literature, commonly combined with the oral administration of a water - soluble contrast medium in the case of esophageal damage, or with angiotomography in the case of vessel damage [4, 5 ]. this diagnostic method in most cases expedites the decision involving the approach method and the extent of the final surgical procedure, provides information on the anatomic ratios of the foreign body to the vital organs, and allows one to foresee the possible consequences of removal of the foreign body. moreover, it can make smaller injuries apparent, which when not discovered can contribute to the development of long - term complications, such as mediastinitis, mediastinum abscess, or dissecting aneurysm of the aortic arch vessels. in the described case, however, the ct scan did not reveal the intraoperatively discovered damage to the outer esophagus wall. we have to remember that patients with retained blades and vessel injuries can be hemodynamically stable on admission, because the blade acts as a local tamponade. unplanned extraction of the blade may result in massive hemorrhage, hemodynamic deterioration and death. sclafani. recommended routine angiography in the case of zone iii neck wounds, whereas atteberry. considered that physical examination alone is sufficient for penetrating zone ii neck trauma [58 ]., that patients should be properly investigated prior to extraction of a retained blade. like other authors, we believe that patients with neck injuries of various etiology should be admitted to multi profile trauma centers, since saving their lives may depend on the cooperation of many different specialists and implementation of multiple diverse therapeutic procedures [2, 3, 6, 9, 10 ]. | we would like to present a case report of a very unusual suicide attempt. a 48-year - old female patient tried to commit suicide by stabbing herself with a kitchen knife into her neck. suicide by self - stabbing is uncommon, constituting only 1 - 3% of suicide attempts. patients with self - inflicted stab wounds may have a higher incidence of surgical interventions based on the stab location. surprisingly, the mortality associated with this kind of wound is low. most medical centers have very limited experience with this infrequent injury. there are only a few case reports and small series published in the literature of the subject. |
cycling extracts were prepared and, while kept on ice, supplemented with demembranated sperm chromatin (to ~400/l extract) and gfp - nls (to ~10 m). extracts were then taken off ice and a portion was loaded into teflon tubing (cole - parmer # yo-06417 - 72), submerged in mineral oil on a glass slide, and cut into 23 mm sections. the tube was imaged at room temperature (2225c) on an inverted epifluorescence microscope (leica dmi6000 b). samples from extract not loaded into the tubes were frozen down and later assayed for histone h1 kinase activity. for studies using pd0166285, extracts were supplemented with a fixed volume of dmso or inhibitor along with the sperm chromatin and gfp - nls. images of the channels were cropped, stitched together, and contrast - adjusted using a custom matlab (mathworks) script. the locations and times at which nuclei underwent envelope breakdown or formation were recorded manually. cycling extracts were prepared and, while kept on ice, supplemented with demembranated sperm chromatin (to ~400/l extract) and gfp - nls (to ~10 m). extracts were then taken off ice and a portion was loaded into teflon tubing (cole - parmer # yo-06417 - 72), submerged in mineral oil on a glass slide, and cut into 23 mm sections. the tube was imaged at room temperature (2225c) on an inverted epifluorescence microscope (leica dmi6000 b). samples from extract not loaded into the tubes were frozen down and later assayed for histone h1 kinase activity. for studies using pd0166285, extracts were supplemented with a fixed volume of dmso or inhibitor along with the sperm chromatin and gfp - nls. images of the channels were cropped, stitched together, and contrast - adjusted using a custom matlab (mathworks) script. the locations and times at which nuclei underwent envelope breakdown or formation were recorded manually. | despite the large size of the xenopus laevis egg (~1.2 mm diameter), a fertilized egg rapidly proceeds through mitosis in a spatially - coordinated fashion. mitosis is initiated by a bistable system of regulatory proteins centered on cdk11,2, raising the possibility that this spatial coordination could be achieved through trigger waves of cdk1 activity3. using an extract system that carries out cell cycles in vitro, we show that mitosis does spread through xenopus cytoplasm via trigger waves, propagating at a linear speed of ~60 m / min. perturbing the feedback loops that give rise to cdk1 s bistability changes the speed and dynamics of the waves. time lapse imaging of intact eggs argues that trigger waves of cdk1 activation are responsible for surface contraction waves, ripples in the cell cortex that precede cytokinesis4,5. these findings indicate that cdk1 trigger waves help ensure the spatiotemporal coordination of mitosis in large eggs. trigger waves may be an important general mechanism for coordinating biochemical events over large distances. |
environmental 18s rdna sequences of opisthokonts were obtained from genbank following a two - step screening process. second, we used these and other published sequences from cultures or environmental surveys that belong to the target groups (but are not labeled as such in genbank) to retrieve additional sequences using blastn. maximum likelihood phylogenetic trees were constructed using raxml with the gtrcati model of evolution and with wide taxon coverage, to establish whether ambiguous divergent sequences belonged to a given group. repeated runs on distinct starting trees were carried out to select the tree with the best topology (the most likely of 1,000 alternative trees). details on the phylogenetic methods are explained elsewhere (del campo and massana 2011). | the opisthokonta clade includes metazoa, fungi, and several unicellular lineages, such as choanoflagellates, filastereans, ichthyosporeans, and nucleariids. to date, studies of the evolutionary diversity of opisthokonts have focused exclusively on metazoans, fungi, and, very recently, choanoflagellates. thus, very little is known about diversity among the filastereans, ichthyosporeans, and nucleariids. to better understand the evolutionary diversity and ecology of the opisthokonts, here we analyze published environmental data from nonfungal unicellular opisthokonts and report 18s ribosomal dna phylogenetic analyses. our data reveal extensive diversity among all unicellular opisthokonts, except for the filastereans. we identify several clades that consist exclusively of environmental sequences, especially among ichthyosporeans and choanoflagellates. moreover, we show that the ichthyosporeans represent a significant percentage of overall unicellular opisthokont diversity, with a greater ecological role in marine environments than previously believed. our results provide a useful phylogenetic framework for future ecological and evolutionary studies of these poorly known lineages. |
obesity and type 2 diabetes (t2d) are two world health concerns of pressing importance, with the prevalence of both increasing at a startling rate. it is estimated by the international diabetes foundation (idf) that the that the global number of adults with type 1 diabetes (t1d) or t2d will grow from 248 million in 2007 to 380 million in 2025.1 currently, in developed countries, 85% to 95% of people with diabetes have t2d, and in developing countries the proportion with t2d (compared with t1d) is higher.1,2 the world health organization (who) estimated that in 2005 there were more than 400 million obese adults, with body mass index (bmi) > 30 kg / m, and 1.6 billion overweight adults.3 according to the latest quality and outcomes framework (qof) estimates, the prevalence of t1d and t2d in england was 3.9% in 2008 (data for t2d alone are not recorded, but are approximately 90% of all cases) and the prevalence of obesity was 7.66% the prevalence of both are increasing each year.4 both t2d5 and obesity6 reduce hrqol. the gravest affect of t2d is due to the macro- and micro - vascular complications which usually develop as the disease progresses.7,8 obese people suffer from impaired hrqol due to specific problems relating to mobility, pain, and/or discomfort,9 but also as a result of increased risk of t2d, coronary heart disease (chd), and hypertension.6,10 obesity and t2d are also closely related, since obesity is the largest risk factor for developing t2d.11 therefore, first - line intervention in the management of t2d includes diet and exercise in an attempt to promote weight loss. unfortunately, however, many of the agents available for the management of t2d often lead to weight gain.12,13 thus knowledge of the relationship between t2d, obesity and hrqol is desirable. several studies have separately investigated weight or diabetes and their effect on hrqol, but the available literature examining the simultaneous effects of weight and diabetes on hrqol is limited.14 cross - sectional data are needed to extend the knowledge in this area. the aim of this study was to evaluate the relationship between obesity, t2d, and hrqol in a real - world setting in the general population of england. we used data collected during the health survey for england (hse) 2003.15 the hse is a series of annual surveys, commissioned by the uk department of health, which covers people living in private households in england. data collection for hse 2003 involved an interview, followed by a visit from a specially trained nurse, and included weight and height measurements, as well as further questioning. each year the hse focuses on a different demographic group and looks at health indicators such as cardiovascular disease, physical activity, eating habits, oral health, accidents, and asthma. the 2003 survey had a primary focus on cardiovascular problems and a secondary focus on diabetes. it also included the eq-5d questionnaire,16 a generic health - related quality - of - life measure. the hse 2003 dataset therefore contained all the parameters for studying the effects of weight, diabetes, confounding illness, and social factors on hrqol. each of the five domains consists of three levels (ie, rated by individuals as no problems, some / moderate problems, or the five domains of the eq-5d are : mobility, self - care, usual activity, pain / discomfort, and anxiety / depression. a lower score is recorded if the individual has lower hrqol, and death is scored as 0.0. values below zero can exist, if in a given situation, death is considered favorable to life in that given health state. to derive utilities, each health state has been assigned a value based on a uk time - trade - off survey.17 during data collection for hse 2003, participants in the survey were asked if they had diabetes, but not whether they had t1d or t2d. a post - hoc interpretation was applied by those collecting the data, whereby if a participant was receiving insulin at the time of interview, and had been diagnosed with diabetes before their 35th birthday, they would be categorized as having t1d. obesity was defined as bmi 30 kg / m and overweight as bmi of 25 to 29.9 kg / m according to the who classification.3,11 the original hse 2003 dataset (18,553 responses) was purged of records from participants that did not receive the eq-5d questionnaire (those < 16 years old), or did not complete it ; who had t1d ; or did not provide measurements needed to calculate bmi. also discarded were : data from participants who failed to inform about smoking status, cardiovascular disease history, blood pressure, history of long standing, or recent acute illnesses, and participants who did not fill out the general health questionnaire (ghq) (which is designed to predict the need for psychological help).18 the ghq is a validated, self - administered, 12-items questionnaire focusing on the inability to carry out normal functions and the appearance of new and distressing phenomena.19 after purging the dataset, the final number of respondents included in the analyses was 12,188, of which 373 (3.1%) had t2d. a multiple linear regression model, consisting of variables describing several factors such as physical and mental well - being, and socio - economic status, and including information on diabetes and bmi, was developed for this study. in the model, hrqol was dependent on socio - economic and health factors and, for analytical purposes, the focus was on variables for weight and diabetes. the dataset was analyzed using multiple linear regression analysis to examine the influence of obesity and t2d on hrqol. age and gender were retained, even if they became statistically insignificant. whether or not all other variables were retained depended on the statistical tests. the interaction between the two disutilities, diabetes and obesity, was analyzed to determine whether being obese and having t2d had a further effect beyond the additive effects of having each condition. the regression model also included variables for socioeconomic status (income), as well as lifestyle indicators affecting health (smoking), and multiple indicators of health status and wellbeing. an acute illness variable described any recent medical history, and a ghq score measured mental wellbeing. comorbidity variables contained various long - term medical conditions that could affect hrqol (such as kidney disease, rheumatism, asthma, back problems, bronchitis, cancer, epilepsy, hearing problems, ulcer, cardiovascular disease, and hypertension ; table 2). when testing for statistical significance, some variables were treated as groups (income, smoking, acute illness, and ghq). t2d was primarily present in participants over 40 years of age, and most frequently in people over 60 years (table 1). approximately 50% of people with t2d were also obese, and normal weight individuals had the lowest prevalence of t2d. the trend of higher t2d prevalence with higher bmi group was consistent across age groups, with the exception of the 1629 year age group, where very small numbers of people with t2d produced inconsistent results, as shown in table 1. in the model, both t2d and obesity had significant, independent effects on hrqol, measured by the eq-5d. the effect of having both conditions is simply the sum of each individual effect (0.056 ; figure 1). compared with people whose income was in the lowest fifth of the population, people with higher incomes tended to have a better hrqol. compared with current smokers, people who had stopped smoking, or never started, had a statistically better hrqol. recent suffering from an acute illness, reduced health - related hrqol, and participants who had a high score on the ghq (indicating mental problems) also had reduced hrqol. having other co - morbidities decreased hrqol, except for hearing problems, which had a positive effect on hrqol. t2d was primarily present in participants over 40 years of age, and most frequently in people over 60 years (table 1). approximately 50% of people with t2d were also obese, and normal weight individuals had the lowest prevalence of t2d. the trend of higher t2d prevalence with higher bmi group was consistent across age groups, with the exception of the 1629 year age group, where very small numbers of people with t2d produced inconsistent results, as shown in table 1. in the model, both t2d and obesity had significant, independent effects on hrqol, measured by the eq-5d. having t2d reduced hrqol by 0.029 points and obesity reduced hrqol by 0.027 points. the effect of having both conditions is simply the sum of each individual effect (0.056 ; figure 1). compared with people whose income was in the lowest fifth of the population, people with higher incomes tended to have a better hrqol. compared with current smokers, people who had stopped smoking, or never started, had a statistically better hrqol. recent suffering from an acute illness, reduced health - related hrqol, and participants who had a high score on the ghq (indicating mental problems) also had reduced hrqol. having other co - morbidities decreased hrqol, except for hearing problems, which had a positive effect on hrqol. also, hrqol decreased as people got older (table 2). this study showed that obese people (bmi 30 kg / m) had a lower hrqol than non - obese people, regardless of whether they have t2d or not. people with t2d, with normal weight (bmi < 25 kg / m), or obesity, had a lower hrqol than those in the same weight groups, without the disease. however, the effect of obesity and t2d is purely additive, with no positive or negative effects of having both conditions at the same time. these results support those from previous investigations of the impact on hrqol of obesity and t2d.20 a study by macran9 that also used hse data (1996) to examine the relationship between weight (bmi) and hrqol (with hrqol quantified according to eq-5d) found a significant difference in hrqol between bmi categories, although results differed by gender. for women, but not men, there was a significant decrease in utility scores with increasing bmi, when adjusted for age and longstanding illness.9 in a study by lee,21 increasing bmi was found to reduce utility in each of the three groups t1d, t2d, and no diabetes. there was no significant difference in the effect of obesity on utility between those with and without diabetes. a recent review of 18 articles investigating the impact of change in body weight on people s utility scores found that utility decreased as body weight increased, regardless of the scoring system used, or the population.14 the review also found that the change in utility score per unit change in bmi, was slightly higher in people with diabetes than in people without diabetes.14 our results support the findings of matza,22 who showed that a 3% higher weight (as a treatment - related attribute) was accompanied by a reduction in hrqol of 0.04 points. the reduction in hrqol associated with having t2d and obesity shown in this study was similar to the impact of aging several decades, or going from having an income in the second highest quintile to being in the lowest quintile. the impact was slightly lower than the impact of a foot ulcer and slightly greater than the impact of a cardiovascular event. although neither t2d nor obesity was correlated with an increase in the risk of problems in the eq-5d sub - model on anxiety (results not shown), people with a high ghq score (ie, people who reported a need for psychological help), had lower hrqol. ghq score could to some extent, be seen as a proxy for anxiety issues stemming from other sources, such as diabetes and obesity. this would support results from the diabetes attitudes wishes and needs (dawn) study, in which people reported they would feel a sense of self - blame if they had to start insulin therapy.23 the idf currently recommends that well - being and psychological status are periodically tested in people with t2d, either by questioning or using a formal instrument.24 the effect of obesity on hrqol in people with t2d should be considered when selecting a therapy. where possible, preference should be given to therapeutic interventions that have a minimal impact on weight gain. in the united kingdom prospective diabetes study (ukpds), weight gain was found to be significantly higher in people with t2d receiving intensive treatment than in those receiving conventional treatment.25 it is recognized that different treatments can have different effects in weight gain biguanides, -glucosidase inhibitors, glp-1 agents, and some insulin analogues are the least likely to cause weight gain and may be associated with weight loss.13,26 the results of the multinational dawn study27 show that that one of the priorities among patients with t2d was the avoidance of weight gain. people with t1d made up almost a quarter of people with diabetes in hse 2003, compared with the european norm of 4% to 5%,28 indicating that the interpretation may have classified too many people as having t1d. however, any choice of age limit as a means of distinguishing between diabetes types will tend to be arbitrary and some degree of misclassification is unavoidable. for the purpose of this article the chosen standard for the hse 2003 was used, but we noted the potential for misclassification and acknowledge that this is one of the limitations of the analysis. however, we do not believe that any misclassification would have had a substantial impact on the overall findings of this analysis. the hse dataset analyzed in the current study did not include people in institutions, which is likely to have resulted in a relatively lower representation of the older population, particularly those with disabilities and severe illness. non - inclusion of this population in our assessment could have led to an underestimation of the mean impact on hrqol of both obesity and diabetes. the prevalence of t2d was only 3.1% in the purged dataset, compared with 3.9% in the uk population in 2003.1 however, it was considered that the remaining people with diabetes were representative of the group as a whole, and therefore, the selection bias would not exaggerate the results in the model. similarly, approximately 34% of the hse 2003 population was excluded, introducing the possibility of non - response bias. however, most people excluded from the analysis were children (< 16 years old ; 58% of those excluded) who did not complete the eq-5d and would not have been classified as t2d patients in our analysis. the results of this study of data from hse 2003 show that having t2d and being obese, as individual conditions, reduce hrqol significantly, but that the effect of having both conditions is purely additive. when we consider that many treatments for t2d cause weight gain, the effect of a treatment - related increase in weight on the hrqol of people with t2d should be taken into account when choosing treatment. in future, longitudinal data may provide information on the effect on hrqol of treatment - associated weight gain on people with t2d. | backgroundweight gain can contribute towards the development of type 2 diabetes (t2d), and some treatments for t2d can lead to weight gain. the aim of this study was to determine whether having t2d and also being obese had a greater or lesser impact on health - related quality of life (hrqol) than having either of the two conditions alone.methodsthe 2003 dataset of the health survey for england (hse) was analyzed using multiple regression analyses to examine the influence of obesity and t2d on hrqol, and to determine whether there was any interaction between these two disutilities.resultst2d reduced hrqol by 0.029 points, and obesity reduced hrqol by 0.027 points. there was no significant interaction effect between t2d and obesity, suggesting that the effect of having both t2d and being obese is simply additive and results in a reduction in hrqol of 0.056.conclusionsbased on analysis of hse 2003 data, people with either t2d or obesity experience significant reduction in hrqol and people with both conditions have a reduction in hrqol equal to the sum of the two independent effects. the effect of obesity on hrqol in people with t2d should be considered when selecting a therapy. |
in 1993, communesin a (1a) was isolated along with communesin b (1b) from a strain of penicillium sp. found growing on a marine alga by the numata group (figure 1). communesins a (1a) and b (1b) exhibit antiproliferative activity against p-388 lymphocytic leukemia cells (ed50 = 3.5 g / ml and 0.45 g / ml, respectively). in addition, communesin b (1b) disrupts actin microfilaments in cultured mammalian cells and shows cytotoxic activity against lovo and kb cells (mic values of 2.0 g / ml and 4.5 g / ml, respectively). several other members of the comunesin family, communesins b h (1b h), were disclosed from related marine fungal strains of penicillium sp. in the following years. with the exception of communesins g (1 g) and h (1h), the communesins show insecticidal activity and antiproliferative activity against a variety of cancer cells, with communesin b (1b) being the most potent. these indole alkaloids contain several interesting structural features including vicinal all - carbon quaternary centers, bis - aminal functionalities, and a complex polycyclic core. the communesins are structurally unique when compared against other known microfilament - disrupting agents, which are primarily macrolides. macrolide microfilament - disrupting agents show considerable structural similarity, and their interactions with actin have been crystallographically characterized, leading to hypotheses regarding their mechanism of action. the unique structure of communesin b (1b) suggests that it may exhibit a novel mechanism of action on the cytoskeleton relative to other microfilament - disrupting agents. the development of a unified synthetic route to the communesins would enable the understanding of their effects on the cellular cytoskeleton while addressing the scarcity of naturally occurring sources of the compounds. communesins (1), nomofungin (2), and perophoramidine (3). in 2001, an intriguing natural product, nomofungin (2) was isolated from an unidentified fungus found on the bark of ficus microcarpa by the hemscheidt group. interestingly, the only structural difference between communesin b (1b) and nomofungin (2) is that communesin b has an aminal moiety instead of the n, o - acetal moiety present in nomofungin. a combination of experimental and theoretical exercises led to the independent discovery by our laboratory and the funk group that the reported structure of nomofungin was incorrect and that it is actually that of communesin b. although the structure of nomofungin was erroneously assigned, its isolation and structural revision to that of an older structure can be viewed as the inception point for all synthetic efforts to the communesin family members over the past decade. interestingly, there were no reports of synthetic efforts toward the communesins from 1993 up to our initial report in 2003. a structurally related compound, the core is comparable to the one found in the communesins, albeit in a higher oxidation state, with the alternate diastereomeric relationship between the vicinal quaternary carbons and without the azepine ring system. perophoramidine (3) possesses modest cytotoxicity against the hct 116 human colon carcinoma cell line (ic50 = 60 m) and induces apoptosis. these complex, polycyclic, bioactive alkaloids have been the subject of intense synthetic efforts over the past decade. numerous approaches have been reported in the literature, including three from our laboratory. herein, we report the evolution of an efficient, unified approach toward the synthesis of these unique alkaloids. our early efforts toward the communesin structure centered on the laboratory implementation of our proposed biosynthesis (scheme 1). as the key step in the process, we envisioned a diels alder cycloaddition to unite the two indole - based fragments by coupling of 5, an n - methylated derivative of the ergot alkaloid aurantioclavine (4), and an o - azaxylylene indolone 6 to generate the bridged lactam 7. we anticipated that lactam 7 would be highly reactive due to the poor alignment of the nitrogen lone pair with the carbonyl. as such, the pendant amino group would be expected to easily open the lactam, thus forming spirocycle 8. further tailoring would produce communesin a (1a) and b (1b). toward this end, ()-aurantioclavine was prepared using known methods, and an enantioselective synthesis of ()-aurantioclavine utilizing our oxidative kinetic resolution (okr) technology was developed. we proceeded to develop an efficient cycloaddition between ()-indole 9 as a model coupling partner and benzyl chloride 10 using conditions previously developed by steinhagen and corey that resulted in a mixture of pentacyclic diastereomers (89% yield). removal of the tosyl group with magnesium in methanol produced a 2:1 mixture of diastereomers 11 and 12 in 80% combined yield, with the desired relative stereochemistry evident in the major diastereomer (cf. 11 and 1a) (scheme 2). despite the success of this model system, more advanced electrophiles (e.g., mesylate 14, cyclopropane 16, or epoxide 17(17)) did not succumb to cycloaddition conditions (scheme 3). nor have we been successful in the oxidation of 11 and 12 at c(8), which would provide a functional handle for introduction of the second quaternary stereocenter. to obviate the difficulties encountered in our attempts to functionalize c(8), we next considered dienes possessing a functional handle at c(8) that could unite diene and dienophile such as benzisoxazole 19, thereby enabling an intramolecular diels thus, when coupled to aurantioclavine 4, benzisoxazole 20 would offer a stable o - methide imine that could react with the indole moiety of compound 19 in a controlled and intramolecular manner. fischer esterification of commercially available carboxylic acid 21 followed by heating in neat sulfuric acid provided the benzisoxazole acid 20 in 44% yield over two steps (scheme 5). treatment of benzisoxazole acid 20 with oxalyl chloride provided the corresponding acid chloride, which was smoothly coupled with aurantioclavine 4 to furnish carboxamide 22 (91% yield, two steps). similarly, 1-methylaurantioclavine 5 reacted with the acid chloride to afford carboxamide 19 (77% yield, two steps). substrates 22 and 19 were subjected to an intramolecular diels alder cycloaddition under acidic conditions. unfortunately, the benzisoxazole reacted with the butenyl side chain of the aurantioclavine core to generate the bridged polycycles 23 and 24. nuclear overhauser effect nmr spectroscopy (noesy) studies and x - ray analysis (figure 2) demonstrated the relative stereochemistry shown for 24 and that of 23 was assigned by analogy. x - ray structure of bridged polycycle 24. at this point, we turned our attention to synthesizing 3-bromooxindole 26, which would be a precursor to an o - methide imine such as reactive intermediate 6, allowing for the construction of the communesin core according to our original biosynthesis - inspired model (scheme 1). aurantioclavine derivative 25 was reacted with bromooxindole 26 in an effort to produce adduct 27 (scheme 6a). interestingly, different reactivity was observed in coordinating and noncoordinating solvents. in thf or acetonitrile, the reaction afforded indole 28 in 69% yield, wherein the oxindole was introduced to position c(2) of the indole nucleus, presumably via rearrangement of the initially formed adduct 27 at c(3) (scheme 6b). sulfonylation of indole 28 with o - nscl under basic conditions was accompanied by unexpected chlorination of the indole moiety to afford chloroindolenine 29 (73% yield), the structure of which was unambiguously confirmed by x - ray crystallography (figure 3). to the best of our knowledge, this constitutes the first use of o - nscl for chlorination of an indole to provide the 3-chloroindolenine. on the other hand, the same coupling of derivatives 25 and 26 in benzene or dichloromethane furnished indole 28 (24% yield) and two additional undesired products 30 (32% yield) and 31 (24% yield) (scheme 6c). adduct 30 results from nucleophilic attack at c(6) of the aurantioclavine indole core, while double adduct 31 is produced from both c(6) and c(2) functionalization. the structure of 30 was unambiguously determined following preparation of lactam 32 (scheme 6d). subjecting 30 to excess sodium hydride and o - nscl conditions functionalized both the oxindole and indole nitrogens (66% yield) and subsequent reduction of the azide allowed for cyclization to lactam 32 in 66% yield. the structure of 32 was confirmed by single - crystal x - ray diffraction (figure 4). x - ray structure of lactam 32. discouraged by the unsuccessful diels alder cycloaddition - based approaches to communesin f (1f), we considered an alternative strategy toward the natural product. in 2007, as a direct result of our efforts toward the communesins and perophoramidine, we developed a method to generate 3,3-disubstituted oxindoles via the base - mediated coupling of oxindole electrophiles with malonate - derived nucleophiles. we also developed an asymmetric variant of this reaction utilizing copper bis(oxazoline) complexes (scheme 7b). with the method shown in scheme 7, we devised a new synthetic strategy that cast our coupling fragments in an umpolung manner, invoking an electrophilic aurantioclavine portion and a nucleophilic right - hand fragment. we first pursued this notion in the context of the model azepine 35 (scheme 8). treatment of 35 with dbu and a pronucleophile (e.g., 36(22) and 38) produced oxindole adducts (i.e., 37 and 39) possessing the key c(7)c(8) linkage in modest, but encouraging yields. importantly, adduct 37 was crystalline, and we confirmed both the new c c bond as well as the relative stereochemistry of the sole diastereomeric isolate via x - ray analysis. having produced the key c(7)c(8) linkage via an umpolung strategy, we treated aurantioclavine - derived bromooxindole 40 with malonate 41 in the presence of dbu (scheme 9). smooth reactivity under our standard conditions led to the isolation of a single stereoisomeric adduct 42 in 74% yield. to our delight, oxindole adduct 42 was amenable to single - crystal x - ray diffraction, however, the x - ray analysis surprisingly revealed that the alkylation occurs with high syn selectivity relative to the existing isobutenyl substituent (figure 5). this result was intriguing, given that in the diels alder cycloaddition of the corresponding indole 9 with the o - azaxylylene derived from benzyl chloride 10, the selectivity at c(7) favored the anti diastereomer 11 (cf. x - ray structure of oxindole adduct 42. since the undesired relative stereochemistry was obtained in adduct 42 from the alkylation of azepine 40 and malonate 41, we explored our strategy in a model system lacking the azepine ring of the oxindole (scheme 10). known silyl ether 43(21,22) was converted into malonate adducts 46 and 47 in 85% and 96% yield, respectively, under our previously reported conditions in scheme 7. importantly, in the nonazepine system, the efficiency of those alkylations is increased, even in these cases where vicinal quaternary centers are generated. methylation of oxindoles 46 and 47 produced 48 and 49 in 99% and 92% yield, respectively. acid - catalyzed desilylation and cyclization of diester 48 proceeded smoothly to furnish lactone 50 in 85% yield as a single diastereomer (scheme 11a). to our delight, lactone 50 underwent decarboxylative allylic alkylation when treated with pd(pph3)4, yielding 51 in 90% yield as a single diastereomer. single - crystal x - ray analysis confirmed that lactone 51 possesses the relative stereochemistry at the vicinal quaternary carbon centers c(7) and c(8) that is needed for further elaboration to communesin f (1f). interestingly, direct decarboxylative allylic alkylation of diester 49 again provided an alkylated product (i.e., 52) as a single diastereomer in 78% yield (scheme 11b). through x - ray analysis, we discovered that the relative stereochemistry at the vicinal quaternary stereocenters c(20) and c(4) of 52 was complementary to that of the lactone 51 and thus ideal for elaboration to perophoramidine (3). at this time, the underlying reasons for the stereochemical relationships observed in these two alkylation reactions are unclear. the fact that the reactions proceed stereodivergently with high diastereocontrol is quite remarkable. work toward building reasonable models for stereoinduction of -quaternary tetrasubstituted enolates in both cyclic and acyclic settings as well as nevertheless, with the promising model systems 51 and 52 completed, we next applied our findings to expedient formal syntheses of communesin f (1f) and perophoramidine (3). as depicted in our retrosynthetic strategy (scheme 12), communesin f could be completed from advanced intermediate 53 in qin s synthesis. we anticipated the initial disconnection of the aminal linkage in 53, thereby revealing oxindole and aniline moieties in 54. we envisioned that the relative stereochemical relationship at c(7) and c(8) of lactone 55 could be established by employing our decarboxylative allylic alkylation. the quaternary center on oxindole 56 was disassembled into 3-bromooxindole 57 and diallyl malonate 44. in the forward synthetic sense, our efforts toward communesin f commenced with the elaboration of 4-bromooxindole 58 to diallyl malonate 60 (scheme 13). treatment of 4-bromoindole 58 with oxalyl chloride and methanol provided an oxoacetate (78% yield, two steps), which was reduced to the corresponding primary alcohol 59 with lialh4 in 91% yield. silylation of the primary alcohol with tipscl (98% yield) and subsequent oxidation with pyridinium tribromide afforded dibromooxindole 57 in 89% yield. despite the extra steric encumbrance of c(4) substitution, we were delighted to find that smooth coupling of dibromooxindole 57 with malonate 44 produced a 3,3-disubstituted oxindole in 95% yield. microwave assisted lactonization of diester 60 with p - tsoh proceeded smoothly to furnish lactone 61 as a single diastereomer (85% yield). gratifyingly, decarboxylative allylic alkylation constructed the quaternary center at c(8) of compound 62 as a single diastereomer in 97% yield under pd(pph3)4 catalysis. the relative stereochemistry at c(7) and c(8) of 61 and 62 was unambiguously confirmed by x - ray analysis. although ozonolysis of alkene 62 delivered aldehyde 63 in 94% yield, attempted reductive amination of aldehyde 63 did not produce the desired lactam 66 (scheme 14). upon treatment of aldehyde 63 with p - methoxybenzylammonium acetate and sodium cyanoborohydride, amine intermediate 64 was likely produced. instead of opening the lactone directly (path a), nucleophilic attack by the newly generated amine at the oxindole moiety (path b), and subsequent ring - shift tautomerization delivered dihydroquinolinone 65 in 67% yield. alternatively, we found that lactam 54 (an analogue of 66) could be obtained via the reaction sequence summarized in scheme 15. the nitro group on compound 62 was reduced to the aniline, which resulted in concomitant lactone ring opening to furnish a bis - oxindole 67 in 80% yield. protection of the primary alcohol with tipscl (90% yield) and protection of the oxindole nitrogen with methyl chloroformate afforded carbamate 68 in 98% yield. ozonolysis of alkene 68 generated aldehyde 69 (94% yield), which underwent subsequent reductive amination and selective lactamization with the electron - deficient oxindole to afford -lactam 54 in 95% yield. with lactam 54 in hand, we envisioned that the piperidine d ring of 70 would be prepared by alh3me2net mediated reductive cyclization (scheme 16). to our disappointment, treatment of lactam 54 with alh3me2net produced undesired pyrrolidinoindoline derivative 71 as a single diastereomer in 61% yield resulting from chemoselective reduction of the n - pmb - lactam in the presence of the oxindole. after cleavage of the tips group by tbaf (98% yield), the pmb group was removed with ddq to provide alcohol 72. the structure of the pentacyclic heterocycle 72 was confirmed by x - ray analysis (figure 6). having failed on our initial exploration, alternative conditions for construction of the piperidine d ring were next explored. treatment of the lactam 54 with lialh4 produced debrominated compound 73 in 83% yield (scheme 17a). x - ray analysis of compound 73 showed a hydrogen - bonding interaction between the carbonyl group of the pmb - protected amide and the nh group of the carbamate. we reasoned that the undesired pyrrolidine was formed preferentially to the piperidine due to the close proximity of the carbamate nh and the carbonyl group of the pmb - protected amide. next, a reductive cyclization reaction was attempted by treatment of 54 with tf2o and nabh4 to construct the piperidine ring. to our surprise, treatment of lactam 54 with tf2o provided the pmb - protected hexacyclic oxindole 76 in 95% yield (scheme 17b). the pmb - protected amide of 54 was activated by tf2o to provide 74, and nucleophilic attack by the aniline functionality furnished pyrrolidinoindoline derivative 75. after the tips group was removed under the reaction conditions, the resultant hydroxyl group attacked the amidinium to generate the propellane structure of hexacyclic oxindole 76. after cleavage of the pmb group using ddq, the propellane structure of hexacyclic oxindole 77 was confirmed using x - ray analysis. despite this unexpected turn of events, we envisaged that the desired aminal 81 could be accessed from the propellane compound 76 using suitable conditions, since the oxidation state at c(9) of 76 is identical to that of the desired aminal 81 (scheme 18). moreover, the reactive n - pmb - pyrrolidinone in 54 is now protected by the propellane structure of 76, thus leaving the oxindole as the only reducible carbonyl group. fortunately, after extensive experimentation, we were pleased to find that reductive cyclization of hexacyclic oxindole 76 could be accomplished with dibal and et2alcl to furnish aminal 81 in 87% yield (scheme 18). presumably, the oxindole of 76 was reduced by dibal to provide 78, and rearrangement of the propellane structure generated iminium 79. after the workup, water attacked the iminium moiety of 79 to afford aniline 80, and the resultant aniline group attacked the iminium of 80 to construct aminal 81. in the last stage of the synthesis, we screened a variety of reaction conditions to remove the pmb group on the lactam 81 (e.g., ddq, can, tfa, etc.), but surprisingly, removal of the pmb group failed under all conditions attempted. this unexpected turn was particularly insidious since the pmb group was easily removed from hexacyclic oxindole 76 by ddq (scheme 17). the cleavage of allyl or benzyl groups were also examined, but disappointingly, cleavage of these groups on the lactam was similarly unsuccessful under several conditions. given the difficulty of removal of pmb, allyl, and benzyl groups, our attention turned to exploring the o - nitrobenzyl group as a protecting group. however, subjecting the hexacyclic oxindole 77 to o - nitrobenzyl bromide under basic conditions to produce the o - nitrobenzyl - protected propellane hexacyclic oxindole turned out to be challenging. thus, we next investigated reductive amination of aldehyde 69 and were pleased to find that treatment of 69 with o - nitrobenzylammonium acetate 82 furnished lactam 83 in 97% yield (scheme 19). formation of the o - nitrobenzyl - protected propellane hexacyclic oxindole using tf2o (75% yield) was followed by reductive cyclization with dibal and et2alcl to furnish aminal 84 in 60% yield. to our delight, we found that removal of the o - nitrobenzyl group could be achieved by photolysis / irradiation at 350 nm in 40% yield. surprisingly, we discovered that removal of the o - nitrobenzyl group to produce compound 53 was also accomplished using 20% aq naoh in methanol at 75 c in 70% yield a previously unknown deprotection protocol. aminal 53 has been advanced by the qin group to communesin f, thus completing our formal synthesis of the natural product. our retrosynthetic analysis of perophoramidine (3) was based on our previously established expedient synthesis of oxindole derivative 52 (scheme 20). we speculated that the aminal and lactam ring functionalities of pentacycle 85, an intermediate in funk s synthesis, could be cleaved, thereby leading to aldehyde 86. c bond of the 6-bromooxindole moiety in 86 was excised to arrive at nitroarene 52. the construction of the contiguous quaternary centers at c(20) and c(4) of allyl ester 52 with the proper relative stereochemistry was accessed by decarboxylative allylic alkylation as previously described (scheme 11b). carbamate 88 was obtained by reduction of nitroarene 52 with titanium chloride and simultaneous oxindole formation to furnish the bis - oxindole moiety 87 in 91% yield followed by protection with boc anhydride in 85% yield. reductive amination of aldehyde 86 with o - nitrobenzylammonium acetate 82 resulted in an amine that underwent in situ lactam formation to afford oxindole lactam 89 in 91% yield (scheme 21) initially, we attempted to generate the o - nitrobenzyl protected propellane hexacyclic oxindole 90 under analogous conditions to those used in our formal synthesis of communesin f on the pseudo - diastereomeric series (vide supra). however, treatment of lactam 89 with tf2o yielded an unexpected azepine 91 in 70% yield (scheme 22). both the boc and the tips groups on amide 89 were removed under the reaction conditions, and the resulting primary alcohol was presumably converted to the corresponding triflate. finally, the aniline likely attacked the newly formed triflate to form azepine 91. after extensive experimentation, we discovered that in contrast to the communesin system, the desired reductive cyclization in the perophoramidine diastereomer occurred directly with alh3me2net to furnish cyclization product 92 in 42% yield (66% yield based on recovered starting material) (scheme 23). the indoline methyl group was converted to a formyl group using pdc oxidation in 62% yield (93% yield based on recovered starting material). to our delight, an attempt to remove the formyl group with 20% aq naoh at 75 c resulted in removal of both the formyl group and the o - nitrobenzyl group to produce aminal 85 in 50% yield. this molecule was previously advanced by the funk group to perophoramidine and constitutes an expedient formal synthesis of the natural product. in conclusion, we have conducted synthetic studies toward unique polycyclic alkaloids and completed formal syntheses of communesin f (1f) in 9% overall yield over 17 steps and perophoramidine (3) in 6% overall yield (13% overall yield, based on recovered starting material) over 10 steps using a unified stereodivergent alkylation approach. the all - carbon quaternary center on the oxindole was established via stabilized enolate alkylation of 3-bromooxindoles, a method previously developed by our laboratory and now shown to be quite versatile even in particularly sterically challenging situations. the complementary relative stereochemistry of the two contiguous quaternary stereogenic centers found in communesin f (1f) and perophoramidine (3), respectively, was established by substrate controlled diastereoselective decarboxylative allylic alkylation. a reductive amination approach furnished the a ring, and reductive cyclization produced the d ring for both communesin f (1f) and perophoramidine (3). en route to the evolution of our eventual successful strategy, we have discovered a method to convert an indole to a 3-chloroindolenine using a mild reagent such as o - nscl during the synthesis. in addition, previously unknown, mild and efficient deprotection conditions for the o - nitrobenzyl group on the lactam were discovered. further studies to rationalize unprecedented complementary selectivity by pd - catalyzed allylic alkylation reactions are currently in progress. to a solution of 4-methyl - n-(2-vinylphenyl)benzenesulfonamide (si-1) (6.11 g, 22.4 mmol, 1.00 equiv) in thf (140 ml) and water (70 ml) were added n - methylmorpholine n - oxide (5.96 g, 50.8 mmol, 2.30 equiv) and osmium tetroxide (11.6 mg, 43.9 mmol, 0.002 equiv). the reaction was concentrated to approximately 50 ml under reduced pressure and then extracted with a mixture ether and thf (1:1) (3 45 ml). the organic layers were dried over sodium sulfate, and the solvent was removed under reduced pressure. impurities were removed by washing solid with dichloromethane to afford diol si-2 (5.43 g, 80% yield) as a white solid : rf = 0.13 (1:1 hexane / etoac) ; h nmr (300 mhz, cdcl3) 8.47 (s, 1h), 7.71 (d, j = 8.3 hz, 2h), 7.39 (dd, j = 8.1, 1.1 hz, 1h), 7.257.18 (m, 3h), 7.157.02 (m, 2h), 4.82 (t, j = 6.5 hz, 1h), 3.663.57 (m, 2h), 2.97 (br, s, 1h), 2.39 (s, 3h), 1.98 (br, s, 1h) ; c nmr (75 mhz, cdcl3) 144.1, 137.1, 136.3, 129.9, 129.8, 129.2, 128.5, 127.4, 127.0, 122.2, 74.78, 66.0, 21.8 ; ir (neat film nacl) 3271, 1318, 1150 cm ; hrms (mm : esi - apci+) m / z calcd for c15h18no4s [m + h ] 308.0951, found 308.0967. to a solution of diol si-2 (500 mg, 1.63 mmol, 1.00 equiv) in toluene (70 ml) was added dibutyltin dimethoxide (410 l, 1.79 mmol, 1.10 equiv). the flask was fitted with a short path distillation apparatus, and approximately half of the solvent was removed by distillation. to this solution were added momcl (136 l, 1.79 mmol, 1.10 equiv) and tetrabutylammonium iodide (900 mg, 2.44 mmol, 1.50 equiv). after addition, the reaction was stirred for 12 h, and then brine was added to this solution. the residue was purified by flash column chromatography (3:1 1:1 hexanes / etoac) to afford alcohol 13 (513 mg, 90% yield, two steps) as a white solid : rf= 0.27 (1:1 hexane / etoac) ; h nmr (300 mhz, cdcl3) 8.76 (s, 1h), 7.72 (d, j = 8.5 hz, 2h), 7.50 (d, j = 8.0 hz, 1h), 7.277.20 (m, 3h), 7.117.02 (m, 2h), 4.834.78 (m, 1h), 4.64 (s, 2h), 3.60 (dd, j = 10.5, 3.5 hz, 1h), 3.483.41 (m, 2h), 3.39 (s, 3h), 2.39 (s, 3h) ; c nmr (75 mhz, cdcl3) 143.9, 137.1, 136.3, 129.7, 129.5, 128.9, 128.2, 127.2, 124.7, 122.0, 97.0, 73.3, 72.4, 55.6, 21.6 ; ir (neat film nacl) 3233, 2932, 1598, 1497, 1335, 1161 cm ; hrms (mm : esi - apci+) m / z calcd for c17h22no5s [m + h ] 352.1213, found 352.1219. a flame - dried flask (25 ml) equipped with a teflon stirbar was charged with sodium hydride (60% dispersion in mineral oil, 22 mg, 0.55 mmol, 1.10 equiv), which was washed 3 times with dry hexanes. then, dmso (5.5 ml) and trimethylsulfoxonium iodide (119 mg, 0.58 mmol, 1.20 equiv) were added. to this solution was added methyl (e)-2-(2-oxoindolin-3-ylidene)acetate (si-3) (100 mg, 0.49 mmol, 1.00 equiv) in a solution of dmso (2.5 ml). after addition, the reaction was stirred for 2 h, and then the temperature was raised to 50 c. brine was added and then the mixture was extracted with etoac (3 5 ml). the combined organic phases were dried over mgso4 and concentrated in vacuo. the residue was purified was by flash column chromatography (3:1 1:1 hexanes / etoac) to afford oxindole 16 as two diastereomers. diastereomer 1 : rf= 0.52 (1:1 hexane / etoac) ; h nmr (300 mhz, cdcl3) 8.28 (br, s, 1h), 7.34 (d, j = 7.7 hz, 1h), 7.22 (dd, j = 7.7, 1.3 hz, 1h), 7.02 (dd, j = 7.7, 1.1 hz, 1h), 6.996.93 (m, 1h), 3.69 (s, 3h), 2.72 (dd, j = 8.6, 7.4 hz, 1h), 2.16 (dd, j = 7.4, 4.5 hz, 1h), 2.04 (dd, j = 8.6, 4.4 hz, 1h) ; c nmr (75 mhz, cdcl3) 177.5, 169.3, 141.8, 127.9, 126.4, 123.0, 122.4, 110.3, 52.4, 34.3, 32.9, 21.1 ; ir (neat film nacl) 3214, 1712, 1622, 1470, 1209 cm ; hrms (mm : esi - apci+) m / z calcd for c12h12no3 [m + h ] 218.0812, found 218.0825. diastereomer 2 : rf= 0.45 (1:1 hexane / etoac) ; h nmr (300 mhz, cdcl3) 7.99 (br, s, 1h), 7.257.19 (m, 1h), 7.02 (td, j = 7.6, 1.0 hz, 1h), 6.93 (d, j = 7.8 hz, 1h), 6.856.80 (m, 1h), 3.75 (s, 3h), 2.66 (t, j = 8.3 hz, 1h), 2.39 (dd, j = 5.0, 8.0 hz, 1h), 1.84 (dd, j = 5.0, 8.6 hz, 1h) ; c nmr (75 mhz, cdcl3) 176.0, 167.7, 141.1, 129.5, 188.0, 122.4, 118.9, 110.3, 52.6, 33.5, 32.9, 21.3 ; ir (neat film nacl) 3256, 1739, 1710 cm ; hrms (mm : esi - apci+) m / z calcd for c12h12no3 [m + h ] 218.0812, found 218.0828. a flame - dried flask (500 ml) equipped with a teflon stirbar was charged with 2-nitrophenylacetic acid 21 (10.0 g, 55.2 mmol, 1.00 equiv), ethanol (60 ml), sulfuric acid (200 l), and toluene (280 ml). the flask was fitted with a condenser, and the solution was refluxed for 14 h. the solvent was removed under reduced pressure and sulfuric acid (280 ml) was added. after addition, the reaction was heated to 110 c and stirred for 90 min. the solution was then poured onto ice (600 g), and the mixture was extracted with ether (3 200 ml). purification was performed via crystallization from water to afford acid 20 (3.94 g, 44% yield, 2 steps) as an off - white solid : h nmr (300 mhz, acetone - d6) 10.82 (br, s, 1h), 7.94 (d, j = 9.0 hz, 1h), 7.75 (d, j = 10.0 hz, 1h), 7.50 (dd, j = 6.5, 9.5 hz, 1h), 7.34 (dd, j = 7.0, 8.5 hz, 1h) ; c nmr (75 mhz, acetone - d6) 158.5, 158.1, 155.3, 132.4, 128.8, 121.4, 121.0, 116.7 ; ir (neat film nacl) 2360, 1731, 1301, 1231, 1189, 753 cm ; hrms (mm : esi - apci+) m / z calcd for c8h6no3 [m + h ] 164.0342, found 164.0341. to a solution of (e)-2-methyl-4-(3-(2-nitroethyl)-1h - indol-4-yl)but-3-en-2-ol (si-4) (386 mg, 1.41 mmol, 1.00 equiv) in thf (14 ml) was added methyl iodide (875 l, 14.1 mmol, 10.0 equiv) at 0 c. sodium hydride (60% dispersion in mineral oil, 562 mg, 14.5 mmol, 10.3 equiv) was then added to the solution, and the mixture was stirred for 25 min at 23 c. the reaction was quenched with satdrated ammonium hydroxide solution and extracted with etoac (3 10 ml). the residue was purified by flash column chromatography (3:1 2:1 hexanes / etoac) to afford (e)-2-methyl-4-(1-methyl-3-(2-nitroethyl)-1h - indol-4-yl)but-3-en-2-ol (si-5) (363.5 mg, 90% yield) as a yellow solid. to a solution of nitro compound si-5 (512 mg, 1.78 mmol, 1.00 equiv) in meoh (125 ml) and 2 n hcl (40 ml) was added amalgamated zinc, which had been formed from zinc dust (6.5 g, 98.3 mmol, 55.0 equiv) and mercuric chloride (1.10 g, 3.55 mmol, 2.00 equiv) in 2 n hcl and subsequently rinsed with meoh. the mixture was stirred at reflux for 3 h. the reaction was then decanted from the remaining amalgam and then basified to ph > 10. the solid was removed by filtration, and the resulting solution was extracted with dichloromethane (3 100 ml). the residue was purified by flash column chromatography (18:1 ch2cl2/meoh) to afford 1-methylaurantioclavine 5 (258 mg, 60% yield) as a yellow oil : rf= 0.30 (18:1 ch2cl2/meoh) ; h nmr (300 mhz, cdcl3) 7.197.12 (m, 2h), 6.896.83 (m, 2h), 5.48 (d, j = 9.0 hz, 1h), 4.92 (d, j = 9.0 hz, 1h), 3.76 (s, 3h), 3.623.54 (m, 1h), 3.133.02 (m, 3h), 2.26 (br, s, 1h), 1.86 (s, 6h) ; c nmr (75 mhz, cdcl3) 138.5, 137.8, 133.3, 127.7, 125.9, 121.1, 117.4, 114.2, 107.3, 62.6, 48.9, 32.7, 30.8, 25.9, 18.4 ; ir (neat film nacl) 3332, 2910, 1554, 1455 cm ; hrms (mm : esi - apci+) m / z calcd for c16h21n2 [m + h ] 241.1699, found 241.1712. to a solution of 2,1-benzisoxazole-3-carboxylic acid (20) (262 mg, 1.60 mmol, 1.25 equiv) in dichloromethane (5 ml) was added oxalyl chloride (420 l, 4.81 mmol, 3.80 equiv) and then a small amount of dmf (20 l). the reaction was stirred for 1 h, and then the solvent was removed under reduced pressure ; the residue was evaporated from benzene (2 ml) to remove excess reagent. dichloromethane (10 ml) and triethylamine (537 l, 3.85 mmol, 3.00 equiv) were added, and to this solution was added aurantioclavine 4 (290 mg, 1.28 mmol, 1.00 equiv). after addition, the reaction was stirred for 60 min, and then brine was added. the residue was purified by flash column chromatography (3:1 1:1 hexanes / etoac) to afford amide 22 (435 mg, 91% yield, 2 steps) as a white solid. (due to the distinct presence of rotameric isomers, the h nmr and c nmr contained extra peaks. see the attached spectrum, supporting information) : h nmr (300 mhz, cdcl3) 9.008.86 (m, 2h), 7.98 (dq, j = 8.9, 0.9 hz, 1h), 7.717.57 (m, 3h), 7.357.27 (m, 2h), 7.257.22 (m, 1h), 7.227.16 (m, 3h), 7.157.07 (m, 2h), 7.077.01 (m, 2h), 7.016.94 (m, 2h), 6.906.83 (m, 2h), 6.70 (d, j = 7.5 hz, 1h), 5.48 (ddq, j = 7.9, 2.8, 1.6 hz, 2h), 4.824.66 (m, 2h), 4.07 (ddd, j = 15.4, 10.0, 5.8 hz, 1h), 3.82 (td, j = 13.0, 2.6 hz, 1h), 3.603.46 (m, 1h), 3.233.12 (m, 3h), 1.96 (d, j = 1.3 hz, 3h), 1.79 (d, j = 1.5 hz, 3h), 1.73 (d, j = 1.6 hz, 2h), 1.64 (d, j = 1.3 hz, 2h) ; c nmr (75 mhz, cdcl3) 158.5, 158.4, 157.9, 157.8, 156.9, 156.8, 137.7, 137.4, 137.2, 136.9, 135.2, 135.1, 131.5, 131.4, 126.4, 126.1, 124.7, 124.3, 123.9, 123.8, 122.1, 121.9, 121.8, 121.7, 121.1, 121.0, 119.9, 118.3, 117.5, 114.9, 113.3, 112.6, 110.1, 109.9, 61.0, 57.5, 44.5, 43.1, 29.1, 25.9, 25.8, 25.7, 18.9, 18.2 ; ir (neat film nacl) 3325, 2914, 2246, 1730, 1616, 1447 cm ; hrms (mm : esi - apci+) m / z calcd for c23h22n3o2 [m + h ] 372.1707, found 372.1721. to a solution of 2,1-benzisoxazole-3-carboxylic acid (20) (37.5 mg, 0.229 mmol, 1.10 equiv) in dichloromethane (500 l) was added oxalyl chloride (59 l, 0.676 mmol, 3.30 equiv) and then a small amount of dmf (1 l). the reaction was stirred for 1 h, and then the solvent was removed under reduced pressure. the residue was evaporated from benzene (1 ml) to remove excess reagent. dichloromethane (1.1 ml) and triethylamine (30 l, 0.215 mmol, 1.03 equiv) were added, and to this solution was added 1-methylaurantioclavine (5) (50.0 mg, 0.208 mmol, 1.00 equiv). after addition, the reaction was stirred for 60 min, and then brine was added. the residue was purified by flash column chromatography (3:1 2:1 hexanes / etoac) to afford amide 19 (61.6 mg, 77% yield, 2 steps) as a white solid : rf= 0.79 (1:2 hexanes / etoac). (due to the distinct presence of rotameric isomers, the h nmr and c nmr contained extra peaks. see the attached spectrum, supporting information) : h nmr (300 mhz, cdcl3) 8.00 (dt, j = 8.9, 1.1 hz, 1h), 7.71 (dt, j = 8.9, 1.1 hz, 1h), 7.61 (ddt, j = 10.9, 9.1, 1.0 hz, 2h), 7.347.26 (m, 3h), 7.247.16 (m, 5h), 7.127.04 (m, 2h), 7.00 (ddd, j = 8.9, 6.4, 0.8 hz, 1h), 6.936.86 (m, 2h), 6.81 (d, j = 1.2 hz, 1h), 6.72 (d, j = 7.5 hz, 1h), 5.50 (ddt, j = 7.4, 2.8, 1.4 hz, 2h), 4.834.64 (m, 2h), 4.134.00 (m, 1h), 3.81 (td, j = 13.0, 2.6 hz, 1h), 3.69 (d, j = 8.6 hz, 6h), 3.643.45 (m, 1h), 3.263.09 (m, 3h), 1.98 (d, j = 1.3 hz, 3h), 1.831.78 (m, 3h), 1.74 (d, j = 1.4 hz, 2h), 1.65 (d, j = 1.3 hz, 2h) ; c nmr (75 mhz, cdcl3) 158.5, 158.5, 157.7, 157.6, 156.9, 156.8, 137.8, 137.7, 137.6, 136.8, 135.7, 135.6, 131.3, 131.2, 126.5, 126.3, 125.9, 124.8, 124.4, 124.3, 124.2, 122.1, 121.6, 121.5, 121.2, 121.1, 120.0, 118.1, 117.3, 115.0, 114.9, 112.3, 111.7, 107.9, 107.7, 60.9, 57.3, 44.5, 43.1, 32.6, 29.0, 25.9, 25.7, 18.9, 18.2 ; ir (neat film nacl) 2913, 2245, 1615, 1455, 1410 cm ; hrms (mm : esi - apci+) m / z calcd for c24h24n3o2 [m + h ] 386.1863, found 386.1877. a flame - dried vial (20 ml) equipped with a teflon stirbar was charged with amide 22 (100 mg, 0.269 mmol, 1.00 equiv) and cooled to 0 c. to this reaction mixture was added a 0.5 m solution of hcl in meoh (2.7 ml, generated from addition of acetyl chloride to methanol at 0 c) at 0 c. the mixture was stirred for 1 h and then warmed to 23 c over 30 min. purification was performed by washing the solid with dichloromethane to afford indole 23 (31.1 mg, 31% yield) as a white solid : rf= 0.22 (1:1 hexane / etoac) ; h nmr (300 mhz, dmso) 11.05 (d, j = 2.5 hz, 1h), 7.357.17 (m, 6h), 7.09 (t, j = 7.7 hz, 1h), 6.61 (dt, j = 7.4, 1.0 hz, 1h), 5.47 (d, j = 6.6 hz, 1h), 4.21 (dt, j = 13.2, 3.7 hz, 1h), 3.45 (td, j = 13.2, 12.5, 2.4 hz, 1h), 3.15 (dt, j = 16.0, 3.0 hz, 1h), 3.082.94 (m, 1h), 2.37 (dd, j = 6.6, 0.9 hz, 1h), 1.77 (s, 3h), 1.03 (s, 3h) ; c nmr (75 mhz, dmso) 164.3, 153.0, 139.6, 137.0, 134.6, 127.3, 126.6, 123.7, 122.6, 121.4, 118.6, 118.0, 115.2, 112.7, 110.2, 96.4, 70.1, 63.1, 61.2, 44.4, 26.9, 26.6, 26.0 ; ir (neat film nacl) 3314, 1681, 753 cm ; hrms (mm : esi - apci+) m / z calcd for c23h22n3o2 [m + h ] 372.1707, found 372.1710. a flame - dried vial (20 ml) equipped with a teflon stirbar was charged with amide 19 (100 mg, 0.259 mmol, 1.00 equiv) and cooled to 0 c. to this solution was added a 0.5 m solution of hcl in meoh (2.6 ml, generated from addition of acetyl chloride to methanol at 0 c) at 0 c. the mixture was stirred for 1 h and then warmed to 23 c over 30 min. purification was performed via flash column chromatography (3:1 1:1 hexanes / etoac) to afford indole 24 (101 mg, 99% yield) as a white solid : rf= 0.29 (1:1 hexane / etoac) ; h nmr (300 mhz, cdcl3) 7.337.32 (m, 1h), 7.247.14 (m, 5h), 7.00 (s, 1h), 6.766.73 (m, 1h), 5.51 (d, j = 6.5 hz, 1h), 4.574.50 (m, 1h), 3.80 (s, 3h), 3.41 (dd, j = 12.4, 10.0 hz, 1h), 3.333.24 (m, 1h), 3.193.12 (m, 1h), 2.57 (d, j = 6.5 hz, 1h), 1.95 (s, 3h), 1.18 (s, 3h) ; c nmr (75 mhz, cdcl3) 165.4, 153.5, 139.8, 137.9, 135.6, 127.5, 127.0, 126.9, 124.6, 121.9, 119.1, 118.4, 115.9, 113.4, 108.4, 97.2, 70.9, 64.1, 61.9, 45.2, 33.0, 27.5, 27.2, 26.6 ; ir (neat film nacl) 3315, 2932, 1699, 1456, 1317, 754 cm ; hrms (mm : esi - apci+) m / z calcd for c24h24n3o2 [m + h ] 386.1863, found 386.1867. to a solution of aurantioclavine 4 (882 mg, 3.90 mmol, 1.00 equiv) in thf (14 ml) were added triethylamine (820 l, 5.88 mmol, 1.50 equiv) and trifluoroacetic anhydride (606 l, 4.29 mmol, 1.10 equiv) at 0 c. purification was performed via flash column chromatography (9:1 3:1 hexanes / etoac) to afford the n - trifluoroacetate - aurantioclavine 25 (1.03 g, 82% yield) as a yellow foam : rf= 0.30 (3:1 hexanes / etoac). (due to the distinct presence of rotameric isomers, the h nmr and c nmr contained extra peaks. see the attached spectrum, supporting information) : h nmr (300 mhz, cdcl3) 8.50 (s, 2h), 7.317.24 (m, 2h), 7.16 (dd, j = 8.1, 7.2 hz, 2h), 7.036.95 (m, 3h), 6.936.86 (m, 2h), 6.23 (d, j = 7.8 hz, 1h), 5.465.31 (m, 2h), 4.39 (dt, j = 13.4, 3.5 hz, 1h), 4.184.06 (m, 1h), 4.053.91 (m, 1h), 3.83 (td, j = 13.2, 2.9 hz, 1h), 3.43 (dddd, j = 17.4, 13.1, 4.2, 1.7 hz, 1h), 3.293.18 (m, 2h), 3.09 (dt, j = 16.5, 2.8 hz, 1h), 1.91 (d, j = 1.3 hz, 3h), 1.86 (d, j = 1.4 hz, 3h), 1.77 (t, j = 1.9 hz, 6h) ; c nmr (75 mhz, cdcl3) 157.5, 157.0, 156.8, 156.3, 138.5, 137.4, 137.2, 137.2, 135.1, 134.4, 124.3, 123.9, 123.5, 123.3, 122.1, 121.9, 121.8, 119.0, 118.8, 118.5, 117.1, 115.2, 115.0, 113.1, 112.6, 110.3, 110.1, 60.7, 60.6, 58.6, 43.7, 43.7, 28.4, 26.2, 25.7, 25.0, 18.9, 18.2 ; ir (neat film nacl) 3361, 2917, 1667, 1441, 1205 cm ; hrms (mm : esi - apci+) m / z calcd for c17h18n2of3 [m + h ] 323.1366, found 323.1365. a flame - dried flask (1000 ml) equipped with a teflon stirbar was charged with 3-(2-azidoethyl)-1h - indole (si-6) (5.03 g, 30.3 mmol, 1.00 equiv), to which were subsequently added thf (150 ml), t - buoh (150 ml), and water (3.75 ml) followed by cooling to 40 c. a 0 c solution of nbs (8.03 g, 45.1 mmol, 1.50 equiv) in thf (450 ml) was then added via cannula over 30 min, and the resulting solution was allowed to warm to 10 c over 2 h. warming continued slowly over 30 min to 0 c. after 20 min at 0 c, the solvent was removed under reduced pressure. purification was performed via flash column chromatography (9:1 1:2 pentanes : ether) to afford bromooxindole 26 (5.46 g, 64% yield) as a yellow solid. 3-(2-azidoethyl)indolin-2-one (si-7) (1.50 g, 25% yield) was also isolated as a light yellow solid. bromooxindole 26 : rf= 0.46 (2:1 hexanes / etoac) ; h nmr (300 mhz, cdcl3) h nmr (300 mhz, cdcl3) 8.12 (br, s, 1h), 7.39 (ddt, j = 7.5, 1.3, 0.6 hz, 1h), 7.31 (td, j = 7.7, 1.3 hz, 1h), 7.12 (td, j = 7.6, 1.0 hz, 1h), 6.93 (d, j = 7.8 hz, 1h), 3.36 (ddd, j = 12.6, 8.1, 5.2 hz, 1h), 3.24 (dt, j = 12.6, 7.6 hz, 1h), 2.77 (ddd, j = 14.2, 8.1, 7.4 hz, 1h), 2.60 (ddd, j = 14.1, 7.8, 5.2 hz, 1h) ; c nmr (75 mhz, cdcl3) 180.5, 141.7, 128.4, 128.1, 123.8, 122.3, 110.2, 48.0, 43.3, 29.5 ; ir (neat film nacl) 3228, 2100, 1693, 1620, 1470 cm ; hrms (mm : esi - apci+) m / z calcd for c10h10n4obr [m + h ] 281.0033, found 281.0040. oxindole si-7 : rf= 0.37 (2:1 hexanes / etoac) ; h nmr (300 mhz, cdcl3) 8.21 (br, s, 1h), 7.23 (d, j = 6.0 hz, 2h), 7.13 (t, j = 7.5 hz, 1h), 6.95 (d, j = 8.0 hz, 1h), 3.413.20 (m, 3h), 2.842.57 (m, 2h) ; c nmr (75 mhz, cdcl3) 176.6, 139.8, 130.6, 129.1, 124.6, 123.5, 111.4, 54.5, 47.6, 38.0 ; ir (neat film nacl) 3252, 2102, 1732, 1619, 1471 cm ; hrms (mm : esi - apci+) m / z calcd for c10h11n4o[m + h ] 203.0927, found 203.0933. a flame - dried vial (20 ml) equipped with a teflon stirbar was charged with indole 25 (120 mg, 0.372 mmol, 1.00 equiv) and bromooxindole 26 (157 mg, 0.559 mmol, 1.50 equiv), which were subsequently dissolved in thf (4 ml). cesium carbonate (243 mg, 0.746 mmol, 2.00 equiv) was then added. after addition, the reaction was stirred for 12 h, and then water was added. the residue was purified by flash column chromatography (9:1 2:1 hexanes / etoac) to afford adduct 28 (134 mg, 69% yield) as a yellow foam : rf= 0.34 (2:1 hexanes / etoac 2 elutions). (due to the distinct presence of rotameric isomers, the h nmr and c nmr contained extra peaks. see the attached spectrum, supporting information) : h nmr (300 mhz, cdcl3) 8.35 (s, 1h), 8.26 (s, 2h), 7.416.95 (m, 12h), 6.916.86 (m, 1h), 6.816.74 (m, 1h), 6.09 (d, j = 7.9 hz, 1h), 5.37 (d, j = 7.9 hz, 1h), 5.335.27 (m, 1h), 4.17 (d, j = 13.8 hz, 1h), 3.95 (d, j = 15.4 hz, 1h), 3.81 (t, j = 13.8 hz, 1h), 3.63 (t, j = 12.2 hz, 1h), 3.333.05 (m, 5h), 3.052.85 (m, 3h), 2.652.51 (m, 2h), 1.83 (d, j = 1.4 hz, 2h), 1.79 (d, j = 1.4 hz, 2h), 1.731.67 (m, 4h), 1.63 (s, 2h) ; c nmr (75 mhz, cdcl3) 178.8, 178.7, 157.1, 156.7, 156.5, 156.0, 141.4, 141.3, 138.4, 137.0, 136.0, 135.6, 135.1, 134.4, 130.1, 129.9, 129.8, 129.5, 129.5, 125.5, 125.0, 124.9, 124.8, 124.4, 123.7, 123.3, 122.4, 122.2, 119.5, 119.0, 118.7, 118.1, 114.9, 111.8, 111.5, 111.2, 111.2, 110.1, 109.8, 60.5, 58.3, 52.7, 52.6, 47.3, 47.3, 43.5, 43.4, 35.0, 34.8, 28.1, 26.3, 25.8, 24.5, 19.0, 18.3 ; ir (neat film nacl) 3335, 2102, 1713, 1674 cm ; hrms (mm : esi - apci+) m / z calcd for c27h26n6o2f3 [m + h ] 523.2064, found 523.2058. to a solution of adduct 28 (183 mg, 0.350 mmol, 1.00 equiv) in thf (4 ml) was added sodium hydride (60% dispersion in mineral oil, 42 mg, 1.05 mmol, 3.00 equiv) at 0 c. the reaction mixture was stirred for 5 min, and o - nitrobenzylsulfonyl chloride (116 mg, 0.523 mmol, 1.50 equiv) was added at 0 c. the reaction was stirred for 10 min, and then a satdrated solution of ammonium chloride was added. the residue was purified by flash column chromatography (15:1 2:1 hexanes / etoac) to afford alkyl chloride 29 (142 mg, 73% yield) as a white crystalline solid : rf= 0.26 (2:1 hexanes / etoac). (due to the distinct presence of rotameric isomers, the h nmr and c nmr contained extra peaks. see the attached spectrum, supporting information) : h nmr (300 mhz, cdcl3) 8.648.59 (m, 2h), 7.957.84 (m, 8h), 7.557.47 (m, 6h), 7.377.26 (m, 5h), 7.15 (d, j = 7.0 hz, 2h), 6.95 (d, j = 8.0 hz, 1h), 6.31 (d, j = 7.5 hz, 2h), 5.765.72 (m, 2h), 5.61 (d, j = 7.5 hz, 1h), 4.204.08 (m, 2h), 3.943.67 (m, 3h), 3.313.19 (m, 2h), 2.992.74 (m, 6h), 2.29 (dd, j = 3.0, 10.5 hz, 1h), 1.75 (s, 6h), 1.65 (s, 6h) ; c nmr (75 mhz, cdcl3) 175.9, 175.8, 173.8, 173.6, 156.3, 155.9, 151.7, 151.6, 148.2, 147.9, 141.6, 140.7, 139.9, 139.7, 139.2, 138.2, 137.4, 136.9, 136.4, 136.0, 135.3, 135.2, 132.6, 130.9, 130.8, 130.4, 130.4, 128.3, 126.8, 126.6, 126.4, 126.3, 125.8, 125.4, 125.3, 125.2, 124.9, 121.7, 121.6, 120.6, 119.8, 118.6, 115.4, 115.3, 114.8, 77.7, 77.4, 77.2, 76.8, 75.7, 75.5, 60.1, 57.6, 55.7, 55.6, 46.5, 46.4, 40.0, 39.1, 37.8, 37.6, 36.1, 32.6, 26.5, 26.0, 18.6, 18.1 ; ir (neat film nacl) 2102, 1755, 1686, 1544, 1146 cm ; hrms (mm : esi - apci+) m / z calcd for c33h28o7n7f3scl [m + h ] 758.1406, found 758.1442. a flame - dried flask (100 ml) equipped with a teflon stirbar was charged with indole 25 (1.03 g, 3.21 mmol, 1.00 equiv) and bromooxindole 26 (2.25 mg, 8.02 mmol, 2.50 equiv), which were subsequently dissolved in dichloromethane (32 ml). cesium carbonate (3.14 g, 9.62 mmol, 3.00 equiv) was then added. after addition, the reaction was stirred for 3 h, and then water was added. the residue was purified by flash column chromatography (18:1 1:2 hexanes / etoac) to afford adduct 28 (404 mg, 24% yield), adduct 30 (538 mg, 32% yield), and adduct 31 (548 mg, 24% yield). (due to the distinct presence of rotameric isomers, the h nmr and c nmr contained extra peaks. see the attached spectrum, supporting information) : h nmr (300 mhz, cdcl3) 8.91 (d, j = 13.0 hz, 2h), 8.33 (d, j = 6.0 hz, 2h), 7.287.03 (m, 8h), 6.956.86 (m, 6h), 6.75 (d, j = 7.5 hz, 1h), 6.11 (d, j = 7.5 hz, 1h), 5.31 (dd, j = 7.5, 29.5 hz, 2h), 4.30 (d, j = 13.5 hz, 1h), 4.064.01 (m, 1h), 3.943.84 (m, 1h), 3.71 (t, j = 13.0 hz, 1h), 3.29 (t, j = 13.0 hz, 1h), 3.173.13 (m, 6h), 2.98 (d, j = 16.5 hz, 1h), 2.892.75 (m, 2h), 2.532.44 (m, 2h), 1.751.69 (m, 10h) ; c nmr (75 mhz, cdcl3) 181.1, 180.9, 157.4, 157.0, 156.7, 156.2, 141.1, 138.5, 137.5, 137.3, 137.1, 135.5, 134.8, 133.4, 133.2, 132.2, 132.1, 128.8, 128.7, 125.1, 124.0, 123.6, 123.2, 123.1, 122.8, 122.5, 119.0, 118.7, 117.0, 115.7, 115.1, 114.9, 113.3, 112.6, 110.7, 108.6, 108.5, 60.6, 58.6, 55.5, 55.4, 47.9, 43.8, 43.7, 36.6, 36.3, 28.4, 26.3, 25.8, 25.0, 18.9, 18.3 ; ir (neat film nacl) 3328, 2100, 1712, 1682 cm. adduct 31 : rf= 0.09 (2:1 hexanes / etoac tmex 2 elutions) ; (due to the distinct presence of rotameric isomers, the h nmr and c nmr contained extra peaks. see the attached spectrum, supporting information) : h nmr (300 mhz, acetone - d6) 10.43 (d, j = 11.7 hz, 1h), 9.80 (d, j = 7.9 hz, 1h), 9.59 (d, j = 4.9 hz, 1h), 7.437.17 (m, 7h), 7.146.92 (m, 6h), 6.67 (dd, j = 18.0, 8.0 hz, 1h), 6.11 (d, j = 8.1 hz, 1h), 5.505.25 (m, 2h), 4.073.48 (m, 4h), 3.343.12 (m, 5h), 3.04 (s, 3h), 2.962.38 (m, 8h), 1.841.58 (m, 7h) ; c nmr (75 mhz, acetone - d6) 180.2, 180.1, 177.9, 177.8, 143.1, 143.0, 142.9, 138.3, 137.4, 137.2, 136.8, 135.3, 134.8, 134.6, 134.4, 133.6, 133.3, 133.2, 133.0, 131.3, 129.9, 129.3, 129.2, 126.1, 125.3, 125.2, 125.0, 124.8, 123.9, 123.6, 122.9, 118.0, 147.1, 111.0, 110.9, 110.3, 109.9, 109.4, 109.2, 61.2, 59.2, 55.8, 55.7, 52.7, 52.7, 48.6, 47.6, 44.0, 37.0, 36.9, 35.4, 28.2, 26.1, 25.7, 24.8, 18.8, 18.3 ; ir (neat film nacl) 3305, 2101, 1713, 1472 cm ; hrms (mm : esi - apci+) m / z calcd for c37h34n10f3o3 [m + h ] 723.2762, found 723.2761. a flame - dried vial (4 ml) equipped with a teflon stirbar was charged with adduct 30 (43.7 mg, 0.0836 mmol, 1.00 equiv), which was subsequently dissolved in thf (800 l). the solution was cooled to 0 c, and then sodium hydride (60% dispersion in mineral oil, 17 mg, 0.425 mmol, 5.00 equiv) was added. five minutes after the sodium hydride addition, o - nitrobenzylsulfonyl chloride (93 mg, 0.420 mmol, 5.02 equiv) was added. the reaction was stirred for 10 min, and then a satdrated solution of ammonium chloride was added. the resulting solution was extracted 3 times with ether, the organic layers were combined and dried over magnesium sulfate, and the solvent was removed under reduced pressure. purification was performed via flash column chromatography (9:1 1:1 hexanes / etoac) to afford the 4-((r)-8-((r)-3-(2-azidoethyl)-1-(2-nitro-4-sulfophenyl)-2-oxoindolin-3-yl)-1-(2-methylprop-1-en-1-yl)-2-(2,2,2-trifluoroacetyl)-1,2,3,4-tetrahydro-6h - azepino[5,4,3-cd]indol-6-yl)-3-nitrobenzenesulfonic acid (si-8) (49.0 mg, 66% yield) as a yellow solid. (due to the distinct presence of rotameric isomers, the h nmr and c nmr contained extra peaks. see the attached spectrum, supporting information) : h nmr (300 mhz, cdcl3) 8.548.51 (m, 2h), 8.038.00 (m, 2h), 7.887.56 (m, 16h), 7.50 (s, 1h), 7.427.35 (m, 5h), 7.187.12 (m, 3h), 6.95 (s, 1h), 6.61 (d, j = 7.5 hz, 1h), 5.97 (d, j = 7.5 hz, 1h), 5.235.16 (m, 2h), 4.27 (d, j = 13.5 hz, 1h), 4.02 (d, j = 15.0 hz, 1h), 3.893.79 (m, 1h), 3.64 (t, j = 11.0 hz, 1h), 3.322.80 (m, 11h), 2.482.33 (m, 2h), 1.73 (s, 12h) ; c nmr (75 mhz, cdcl3) 176.2, 176.0, 157.3, 156.9, 156.5, 148.0, 148.0, 140.2, 140.1, 140.0, 138.6, 137.2, 136.6, 136.2, 136.0, 135.7, 135.5, 134.8, 134.7, 132.8, 132.8, 132.4, 131.2, 131.2, 131.1, 130.9, 130.6, 129.9, 129.8, 129.5, 129.2, 127.4, 127.0, 125.7, 125.7, 125.4, 125.4, 125.2, 125.1, 124.9, 122.8, 121.9, 121.1, 120.0, 118.8, 118.5, 117.9, 115.5, 114.9, 110.6, 60.2, 58.1, 55.0, 55.0, 47.5, 42.8, 42.7, 36.6, 36.6, 39.9, 28.0, 26.3, 25.8, 25.0, 18.9, 18.3 ; ir (neat film nacl) 2930, 2101, 1754, 1684, 1544 cm. to a solution of nosylate si-8 (43.1 mg, 0.0483 mmol, 1.00 equiv) in thf (1 ml) and water (250 l) was added triphenylphosphine (25 mg, 0.0953 mmol, 2.00 equiv). the reaction was stirred for 3 h at 50 c, and then the solvent was removed under reduced pressure. the residue was purified by flash column chromatography (3:1 1:1 hexanes / etoac) to afford the lactam 32 (49.0 mg, 66% yield) as a yellow crystalline solid : rf= 0.14 (1:1 hexanes / etoac). (due to the distinct presence of rotameric isomers, the h nmr and c nmr contained extra peaks. see the attached spectrum, supporting information) : h nmr (300 mhz, cdcl3) 10.82 (s, 2h), 7.757.50 (m, 17h), 7.41 (td, j = 7.8, 1.4 hz, 2h), 7.367.23 (m, 9h), 7.13 (dd, j = 7.5, 1.9 hz, 1h), 6.80 (dd, j = 6.8, 1.6 hz, 2h), 6.54 (d, j = 7.6 hz, 1h), 5.99 (d, j = 7.7 hz, 1h), 5.345.16 (m, 2h), 4.26 (d, j = 13.4 hz, 1h), 4.00 (d, j = 15.6 hz, 1h), 3.80 (dd, j = 14.3, 10.2 hz, 1h), 3.683.48 (m, 3h), 3.322.97 (m, 9h), 2.29 (q, j = 7.8, 6.7 hz, 2h), 1.66 (d, j = 1.3 hz, 6h), 1.60 (s, 6h) ; c nmr (75 mhz, cdcl3) 180.0, 157.3, 156.8, 156.7, 156.2, 147.9, 147.8, 147.7, 139.5, 139.4, 137.8, 137.5, 137.4, 136.9, 136.5, 136.4, 136.2, 136.1, 135.6, 135.3, 135.1, 133.6, 132.9, 132.5, 132.2, 132.0, 131.0, 130.9, 130.5, 129.0, 127.9, 127.8, 126.3, 126.2, 125.2, 125.1, 125.0, 124.9, 124.8, 124.5, 124.4, 124.3, 123.3, 122.4, 121.4, 120.5, 119.2, 118.9, 118.5, 118.4, 115.1, 110.2, 109.7, 77.7, 77.4, 77.2, 76.8, 60.1, 58.2, 57.7, 43.0, 42.8, 39.8, 38.8, 28.5, 26.1, 25.7, 25.3, 18.9, 18.3 ; ir (neat film nacl) 3098, 2916, 1682, 1545, 1368, 1170, 732 cm ; hrms (mm : esi - apci+) m / z calcd for c39h34n6o10f3s2 [m + h ] 867.1729, found 867.1735. to a solution of 2,3,4,6-tetrahydro-1h - azepino[5,4,3-cd]indole (si-9) (106 mg, 0.614 mmol, 1.00 equiv) and et3n (0.17 ml, 1.23 mmol, 2.00 equiv) in ch2cl2 (4 ml) cooled to 0 c was added a solution of tscl (117 mg, 0.614 mmol, 1.00 equiv) in ch2cl2 (3 ml) dropwise. the ice bath was removed, and the reaction mixture was stirred for 5 h, diluted with etoac (160 ml), and washed with 0.5 n hcl (2 30 ml) and brine. the organic layers were combined, dried over mgso4, and concentrated in vacuo. the residue was purified by column chromatography (2:1 hexanes / etoac) to afford 2-tosyl-2,3,4,6-tetrahydro-1h - azepino[5,4,3-cd]indole (si-10) (164 mg, 82% yield). indole si-10 was dissolved in thf (10 ml), t - buoh (10 ml), and water (1 ml). the solution was cooled to 0 c, and pyridinium tribromide (504 mg, 1.54 mmol, 1.02 equiv) was added. the reaction mixture was stirred at 0 c for 45 min and then allowed to warm to ambient temperature. the reaction was quenched by addition of 10 ml of 1:1 v / v 1 m na2s2o3/satd nahco3. the reaction mixture was diluted with brine (50 ml) and extracted with etoac (3 50 ml). the residue was purified by silica gel chromatography (2:1 hexanes / acetone) to afford 2-tosyl-1,2,3,4,4a,6-hexahydro-5h - azepino[5,4,3-cd]indol-5-one (si-11) (397 mg, 75% yield) as a white solid : rf= 0.15 (1:1 hexanes / etoac) ; h nmr (300 mhz, cdcl3) 8.77 (br, s, 1h), 7.56 (d, j = 8.2 hz, 2h), 7.217.14 (m, 3h), 6.94 (d, j = 7.7 hz, 1h), 6.80 (d, j = 7.7 hz, 1h), 4.80 (d, j = 15.5 hz, 1h), 4.30 (d, j = 13.6 hz, 1h), 4.15 (d, j = 15.5 hz, 1h), 3.52 (dd, j = 12.5, 3.5 hz, 1h), 3.24 (t, j = 12.6 hz, 1h), 2.38 (s, 3h), 2.30 (m, 1h), 1.53 (m, 1h) ; c (75 mhz, cdcl3) 178.4, 143.3, 140.5, 137.1, 135.9, 129.6, 128.4, 128.3, 127.0, 121.5, 109.1, 53.3, 51.6, 46.2, 28.6, 21.5 ; ir (neat film nacl) 3276, 2925, 2853, 1698, 1618, 1463, 1326, 1153 cm ; hrms (mm : esi - apci+) m / z calcd for c18h19n2o3s [m + h ] 343.1111, found 343.1104. lihmds (429 mg, 2.57 mmol, 2.50 equiv) was dissolved in thf (5 ml). the solution was cooled to 78 c, and a solution of oxindole si-11 (352 mg, 1.03 mmol, 1.00 equiv) in thf (20 ml) was added dropwise over 20 min. the reaction mixture was stirred at 78 c for 20 min and transferred to a precooled solution of n - bromosuccinimide (457 mg, 2.57 mmol, 2.50 equiv) in thf (10 ml) that was protected from light. the resulting reaction mixture was placed in a 40 c bath for 1 h, while being protected from light, and then quenched with satd nh4cl. the reaction mixture was allowed to warm to ambient temperature, diluted with brine (100 ml), and extracted with etoac (3 70 ml). the combined organic extracts were dried over mgso4 and concentrated to afford a yellow oil, which was purified by silica gel chromatography (2:1 hexanes / etoac) to afford bromooxindole 35 (334 mg, 76% yield) as a yellow solid : rf = 0.40 (1:1 hexane / etoac) ; h nmr (300 mhz, cdcl3) 8.49 (br, s, 1h), 7.58 (d, j = 8.2 hz, 2h), 7.247.19 (m, 3h), 6.96 (d, j = 7.7 hz, 1h), 6.83 (d, j = 7.8 hz, 1h), 4.76 (d, j = 15.4 hz, 1h), 4.32 (m, 2h), 3.80 (t, j = 13.2 hz, 1h), 2.39 (s, 3h), 2.33 (m, 1h), 1.90 (m, 1h) ; c nmr (75 mhz, cdcl3) 175.1, 143.5, 139.0, 137.7, 136.9, 130.7, 129.7, 128.7, 126.9, 122.6, 110.1, 59.2, 51.6, 48.3, 35.2, 21.5 ; ir (neat film nacl) 3313, 2930, 1734, 1615, 1460, 1334, 1155, 1096, 727 cm ; hrms (mm : esi - apci+) m / z calcd for c18h18brn2o3s [m + h ] 421.0216, found 421.0213. a solution of bromooxindole 35 (44.9 mg, 0.107 mmol, 1.00 equiv) and 2-nitrophenylacetonitrile 36 (34.6 mg, 0.213 mmol, 2.00 equiv) in thf (3 ml) was cooled to 78 c. dbu (64 ml, 0.426 mmol, 4.00 equiv) was added dropwise. after 8 h, the reaction mixture was quenched with satd nh4cl (10 ml) and the mixture was extracted with etoac (4 5 ml). the combined organic extracts were dried over mgso4 and concentrated under reduced pressure to afford a brown oil, which was purified by preparatory thin - layer chromatography on silica gel (1:1 hexane / etoac x2 elutions) to afford nitrile 37 (34.8 mg, 64% yield) as an orange - yellow solid : rf = 0.28 (1:1 hexane / etoac) ; h nmr (300 mhz, cdcl3) 7.92 (m, 1h), 7.63 (m, 2h), 7.55 (m, 2h), 7.43 (m, 1h), 7.29 (d, j = 8.0 h, 2h), 7.18 (t, j = 7.8 hz, 1h), 6.97 (d, j = 7.7 hz, 1h), 6.48 (d, j = 7.4 hz, 1h), 6.07 (s, 1h), 4.80 (d, j = 16.2 hz, 1h), 4.29 (m, 2h), 3.45 (app. dd, j = 15.0, 2.6 hz, 1h), 2.43 (s, 3h), 1.94 (m, 1h) ; c nmr (75 mhz, cdcl3) 176.2, 147.9, 143.8, 140.3, 137.9, 136.5, 133.5, 132.5, 131.0, 130.3, 130.0, 127.0, 124.8, 124.5, 124.5, 123.7, 116.2, 109.4, 54.4, 52.6, 46.8, 34.2, 32.1, 21.6 ; ir (neat film nacl) 3302, 2920, 1724, 1527, 1155, 724 cm ; hrms (mm : esi - apci+) m / z calcd for c26h23n4o5s [m + h ] 503.1384, found 503.1411. to a solution of bromooxindole 35 (50.0 mg, 0.119 mmol, 1.00 equiv) and malonate 38 (90.1 mg, 0.356 mmol, 3.00 equiv) in thf (0.6 ml) was added dbu (54.2 mg, 0.356 mmol, 3.00 equiv) at 78 c. the mixture was extracted with etoac (3 1 ml) and brine. the combined organic extracts were dried over mgso4 and concentrated under reduced pressure, and then the residue was purified by preparatory thin - layer chromatography on silica gel (1:1 hexane / etoac) to afford nitrile 39 (23 mg, 32% yield) : rf = 0.15 (1:1 hexanes / etoac) ; h nmr (300 mhz, cdcl3) 8.85 (br, s, 1h), 8.23 (dd, j = 8.0, 1.5 hz, 1h), 7.587.51 (m, 5h), 7.24 (d, j = 8.0 hz, 2h), 6.96 (dd, j = 7.5, 1.5 hz, 1h), 6.76 (d, j = 1.5 hz, 1h), 6.66 (d, j = 1.0 hz, 1h), 4.44 (d, j = 15.4 hz, 1h), 4.15 (d, j = 15.4 hz, 1h), 3.95 (d, j = 14.6 hz, 1h), 3.82 (s, 3h), 3.75 (s, 3h), 3.69 (m, 1h), 2.40 (s, 3h), 1.95 (d, j = 14.6 hz, 1h), 1.66 (m, 1h) ; c nmr (75 mhz, cdcl3) 179.1, 168.8, 148.1, 143.4, 141.2, 138.7, 137.0, 134.7, 133.9, 132.0, 129.7, 129.1, 128.3, 126.8, 125.7, 122.8, 110.7, 74.8, 68.7, 53.7, 53.5, 51.3, 46.0, 33.4, 21.5 ; ir (neat film nacl) 3313, 1737, 1623, 1530, 1450, 1347, 1241, 1153, 1091, 895, 729 cm ; hrms (fab) m / z calcd for c29h26n3o9s [m h ] 592.1395, found 592.1382. to a solution of aurantioclavine 4 (300 mg, 0.00133 mol, 1.00 equiv) and et3n (0.37 ml, 0.00265 mol, 2.00 equiv) in ch2cl2 (4 ml) cooled to 0 c was added a solution of tscl (254 mg, 0.00133 mmol, 1.00 equiv) in ch2cl2 (3 ml) dropwise. the ice bath was removed, and the reaction mixture was stirred for 5 h, then diluted with etoac (200 ml) and washed with 0.5 n hcl (2 35 ml) and brine. the organic layers were combined, dried over mgso4 and concentrated in vacuo. the residue was purified by column chromatography (2:1 hexanes / etoac) to afford 1-(2-methylprop-1-en-1-yl)-2-tosyl-2,3,4,6-tetrahydro-1h - azepino[5,4,3-cd]indole (si-12) (405 mg, 80% yield). a solution of indole si-12 (902.2 mg, 2.371 mmol, 1.00 equiv) in thf / t - buoh / h2o (10:10:1 v / v / v, 52.5 ml) was cooled to 0 c, and pyridinium tribromide (834.2 mg, 2.608 mmol, 1.10 equiv) was added in small portions over 5 min. the reaction mixture was stirred at 0 c for 15 min, and then allowed to warm to ambient temperature. after 5 min at ambient temperature, the reaction mixture was quenched by addition of 1:1 v / v satd nahco3/1 m aq na2s2o3 (15 ml), poured into brine (150 ml), and extracted with etoac (3 100 ml). the combined extracts were dried over na2so4 and concentrated under reduced pressure to afford a brown solid, which was purified by silica gel chromatography (2:1 1:1 hexanes / etoac) to afford 1-(2-methylprop-1-en-1-yl)-2-tosyl-1,2,3,4,4a,6-hexahydro-5h - azepino[5,4,3-cd]indol-5-one (si-13) (747.5 mg, 80% yield) : rf = 0.22 (5:1 benzene / mecn) ; h nmr (300 mhz, cdcl3) 8.12 (br, s, 1h), 7.33 (dd, j = 8.5, 2.1 hz, 2h), 7.03 (m, 1h), 6.96 (d, j = 7.2 hz, 2h), 6.82 (d, j = 7.7 hz, 1h), 6.64 (d, j = 8.0 hz, 1h), 5.76 (d, j = 8.0 hz, 1h), 5.31 (m, 1h), 4.00 (dt, j = 15.7, 2.8 hz, 1h), 3.643.50 (m, 2h), 2.22 (s, 3h), 2.00 (m, 1h), 1.67 (s, 3h), 1.65 (s, 3h), 1.25 (m, 1h) ; c nmr (75 mhz, cdcl3) 178.4, 142.9, 140.9, 140.5, 138.2, 129.2, 128.3, 128.3, 127.0, 126.7, 121.3, 119.0, 108.7, 59.0, 46.0, 43.8, 27.8, 26.0, 21.4, 18.5 ; ir (neat film nacl) 3246, 2925, 1713, 1615, 1460, 1326, 1155, 732 cm ; hrms (mm : esi - apci+) m / z calcd for c22h25n2o3s [m + h ] 397.1580, found 397.1586. a solution of oxindole si-13 (172.0 mg, 0.434 mmol, 1.00 equiv) in thf (5 ml) was added dropwise to a freshly prepared solution of lihmds (217.8 mg, 1.301 mmol, 3.00 equiv) in thf (5 ml) that had been precooled to 78 c. after 20 min at 78 c, the resulting solution was transferred via cannula to a solution of n - bromosuccinimide (231.6 mg, 1.301 mmol, 3.00 equiv) in thf (5 ml) that had been precooled to 78 c. the resulting yellow reaction mixture was allowed to warm to 15 c (the reaction flask was transferred to a bath composed of ethylene glycol and dry ice) and maintained at this temperature for 2 h. the reaction mixture was then cooled to 78 c and quenched by addition of satd nh4cl (5 ml). the yellow reaction mixture was allowed to warm to ambient temperature and diluted with h2o (80 ml), then extracted with etoac (3 70 ml). the combined organic extracts were washed with brine (100 ml), dried over mgso4, and concentrated under reduced pressure to afford a yellow oil, which was purified immediately by silica gel chromatography (2:1 hexanes / etoac) to afford a 3:1 mixture of bromooxindole 40 (> 20:1 dr) and dehydrobromination product, 1-(2-methylprop-1-en-1-yl)-2-tosyl-1,2,3,6-tetrahydro-5h - azepino[5,4,3-cd]indol-5-one (si-14) (131.1 mg, 64% combined yield, 50% yield of bromooxindole 40). the relative configuration of this bromooxindole was assigned based on the stereochemistry of the malonate adduct obtained (see below). quenching the reaction prior to completion afforded the bromooxindole 40 as a single diastereomer, which showed greater stability, and could be fully characterized : rf = 0.50 (1:1 hexane / etoac) ; h nmr (300 mhz, cdcl3) 8.62 (br, s, 1h), 7.49 (d, j = 8.0 hz, 2h), 7.21 (t, j = 8.0, 1h), 7.13 (d, j = 8.0 hz, 2h), 6.90 (d, j = 7.5 hz, 1h), 6.79 (dd, j = 8.0, 1.0 hz, 1h), 5.96 (td, j = 8.5, 1.5 hz, 1h), 5.88 (d, j = 8.5 hz, 1h), 4.184.02 (m, 2h), 2.35 (s, 3h), 2.27 (m, 1h), 1.97 (m, 1h), 1.76 (d, j = 1.0 hz, 3h), 1.75 (d, j = 1.5 hz, 3h) ; c nmr (75 mhz, cdcl3) 174.8, 143.2, 142.4, 140.2, 139.2, 137.8, 130.8, 129.4, 126.9, 126.1, 122.7, 120.1, 109.7, 66.8, 59.5, 39.8, 34.4, 26.1, 21.4, 18.4 ; ir (neat film nacl) 3291, 1734, 1617, 1602, 1457, 1326, 1156, 1092, 738 cm ; hrms (mm : esi - apci+) m / z calcd for c22h24brn2o3s [m + h ] 475.0686, found 475.0668. bromooxindole 40 (> 20:1 dr, 51.3 mg, 0.108 mmol, 1.00 equiv) was dissolved in thf (2 ml). dimethyl malonate 41 (37 ml, 0.324 mmol, 3.00 equiv) was added, and the reaction mixture was cooled to 78 c. dbu (48 ml, 0.324 mmol, 3.00 equiv) was added dropwise. the reaction mixture was stirred at 78 c for 15 min and then warmed to 23 c. after the reaction mixture was maintained at 23 c for 6 h, satd nh4cl (2 ml) was added and the mixture was warmed to ambient temperature. the reaction mixture was diluted with etoac (50 ml) and satd nh4cl (50 ml). the phases were separated, and the aqueous phase was extracted with etoac (2 50 ml). the combined organic extracts were dried over na2so4 and concentrated to afford colorless oil. analysis of the crude oil by h nmr indicated > 20:1 dr of the malonate adduct 42. the residue was purified by silica gel chromatography (1:1 hexane / etoac) to afford malonate 42 (42 mg, 74% yield) : rf = 0.20 (1:1 hexane / etoac) ; h nmr (300 mhz, cdcl3) 7.88 (br, s, 1h), 7.26 (d, j = 8.0 hz, 2h), 7.14 (t, j = 7.5 hz, 1h), 7.00 (d, j = 8.0 hz, 2h), 6.91, (d, j = 7.5 hz, 1h), 6.72 (d, j = 8.0 hz, 1h), 5.92 (d, j = 4.5 hz, 1h), 5.38 (d, j = 4.5 hz, 1h), 4.62 (s, 1h), 4.06 (m, 1h), 3.89 (m, 1h), 3.78 (s, 3h), 3.46 (s, 3h), 2.40 (m, 1h), 2.30 (s, 3h), 1.82 (s, 3h), 1.79 (s, 3h), 1.07 (m, 1h) ; c nmr (75 mhz, cdcl3) 177.8, 166.8, 166.8, 143.0, 141.9, 141.4, 141.0, 137.9, 129.2, 128.9, 127.6, 127.0, 123.0, 124.0, 109.0, 59.6, 53.9, 52.9, 52.3, 51.3, 39.8, 28.9, 26.4, 21.4, 18.6 ; ir (neat film nacl) 3338, 2953, 1733, 1618, 1597, 1458, 1327, 1158 cm ; hrms (mm : esi - apci+) m / z calcd for c27h31n2o7s [m + h ] 527.1846, found 527.1848. to a suspension of cs2co3 (5.37 g, 16.5 mmol, 3.00 equiv) and bromooxindole 43 (2.27 g, 5.50 mmol, 1.00 equiv) in thf (100 ml) was added malonate 44 (5.04 g, 16.5 mmol, 3.00 equiv) at 0 c. the reaction mixture was then allowed to slowly warm to 23 c and stirred for 16 h. solids were removed via filtration through a celite plug (rinsed with etoac), and the resulting purple solution was concentrated under reduced pressure. the residue was purified by column chromatography using a teledyne isco combiflash (sio2, 120 g column, 100:0 3:1 hexanes / etoac) to provide alkylation product 46 (8.9 g, 85% yield) as a colorless oil : rf = 0.18 (3:1 hexanes / etoac) ; h nmr (500 mhz, cdcl3) 7.97 (d, j = 8.0 hz, 1h), 7.94 (s, 1h), 7.73 (dd, j = 7.9, 1.6 hz, 1h), 7.41 (dt, j = 8.0, 1.6 hz, 1h), 7.387.33 (m, 2h), 7.13 (dt, j = 7.7, 1.1 hz, 1h), 6.89 (dt, j = 7.7, 1.0 hz, 1h), 6.72 (d, j = 7.6 hz, 1h), 5.885.73 (m, 2h), 5.25 (ddd, j = 17.2, 2.8, 1.4 hz, 1h), 5.195.11 (m, 3h), 4.69 (tdd, j = 13.3, 5.7, 1.4 hz, 1h), 4.64 (tdd, j = 13.3, 5.7, 1.3 hz, 1h), 4.57 (tdd, j = 13.4, 5.9, 1.4 hz, 1h), 4.48 (tdd, j = 13.1, 5.9, 1.1 hz, 1h), 3.35 (ddd, j = 9.5, 8.5, 6.9 hz, 1h), 3.07 (dt, j = 9.5, 4.5 hz, 1h), 2.95 (ddd, j = 12.9, 8.7, 7.0 hz, 1h), 2.63 (ddd, j = 12.9, 8.5, 4.5 hz, 1h), 0.970.77 (m, 21h) ; c nmr (125 mhz, cdcl3) 177.8, 166.7, 166.3, 150.2, 140.7, 132.4, 131.3, 131.0, 130.8, 129.5, 129.2, 128.5, 126.9, 125.3, 122.4, 119.2, 118.4, 109.1, 66.8, 66.7, 59.5, 56.7, 38.3, 17.8, 11.8 ; ir (neat film nacl) 3332, 2942, 1714, 1649, 1618, 1538, 1471, 1356, 1230, 1114, 995, 933, 885, 850, 752, 683 cm ; hrms (mm : esi - apci+) m / z calcd for c34h45n2o8si [m + h ] 637.2940, found 637.2945. to a suspension of oxindole 46 (0.50 g, 0.79 mmol, 1.00 equiv) and cs2co3 (0.77 g, 2.37 mmol, 3.00 equiv) in thf (4.0 ml) was added methyl iodide (0.3 ml, 4.7 mmol, 6.00 equiv) at 0 c. the reaction mixture was stirred for 12 h at 23 c. after the reaction was complete, satd nh4cl was added. the combined organic phases were washed with brine, dried over mgso4, and concentrated in vacuo. the residue was purified by flash column chromatography on silica gel (3:1 hexanes / etoac) to give methylated oxindole 48 (0.51 g, 99% yield) : rf = 0.33 (3:1 hexanes / etoac) ; h nmr (500 mhz, cdcl3) 7.82 (d, j = 8.0 hz, 1h), 7.54 (dd, j = 7.9, 1.5 hz, 1h), 7.42 (d, j = 7.6 hz, 1h), 7.31 (dt, j = 7.7, 1.6 hz, 1h), 7.25 (dt, j = 7.8, 1.2 hz, 1h), 7.10 (dt, j = 7.7, 0.9 hz, 1h), 6.85 (dt, j = 7.8, 0.8 hz, 1h), 6.57 (d, j = 7.7 hz, 1h), 5.80 (tdd, j = 16.3, 10.7, 5.8 hz, 1h), 5.71 (tdd, j = 16.4, 10.5, 5.9 hz, 1h), 5.225.02 (m, 4h), 4.694.56 (m, 2h), 4.52 (tdd, j = 13.1, 5.8, 1.3 hz, 1h), 4.36 (tdd, j = 13.3, 5.9, 1.3 hz, 1h), 3.153.02 (m, 4h), 2.96 (ddd, j = 9.7, 8.3, 4.7 hz, 1h), 2.85 (td, j = 13.2, 7.4 hz, 1h), 2.67 (ddd, j = 12.8, 8.0, 4.7 hz, 1h), 0.860.71 (m, 21h) ; c nmr (125 mhz, cdcl3) 176.3, 166.5, 166.5, 150.3, 143.6, 132.7, 131.3, 130.9, 130.3, 128.8, 128.5, 128.5, 128.3, 126.9, 125.1, 122.3, 119.1, 118.5, 107.3, 66.7, 66.7, 59.6, 56.8, 37.9, 26.1, 17.8, 11.7 ; ir (neat film nacl) 3421, 3054, 2944, 2866, 1723, 1613, 1539, 1473, 1356, 1253, 1180, 1104, 1068, 935, 862, 840, 752, 690 cm ; hrms (mm : esi - apci+) m / z calcd for c35h47n2o8si [m + h ] 651.3096, found 651.3092. acetyl chloride (46.0 l, 650 mol, 10.0 equiv) was added to meoh (1.0 ml) and cooled to 0 c. the resulting mixture was stirred for 30 min, and then the solution of oxindole 48 (21.0 mg, 32.0 mol, 1.00 equiv) in meoh (2.0 ml) was added. the reaction was stirred for 2 h at 23 c and then heated to 65 c. the colorless solution was cooled to ambient temperature, concentrated under reduced pressure, and subjected to column chromatography (4:1 hexanes / etoac) to afford the desired lactone 50 (12 mg, 85% yield) as a colorless solid : rf = 0.29 (50% etoac in hexanes) ; h nmr (400 mhz, cdcl3) 7.80 (d, j = 7.4 hz, 1h), 7.397.31 (m, 3h), 7.237.15 (m, 2h), 6.826.75 (m, 2h), 5.92 (tdd, j = 17.1, 10.6, 5.8 hz, 1h), 5.30 (ddd, j = 17.2, 2.7, 1.3 hz, 1h), 5.21 (ddd, j = 10.4, 2.4, 1.2 hz, 1h), 4.984.90 (m, 2h), 4.74 (tdd, j = 13.1, 5.8, 1.3 hz, 1h), 4.714.64 (m, 1h), 3.32 (s, 3h), 2.842.70 (m, 1h), 2.03 (m, 1h) ; c nmr (100 mhz, cdcl3) 175.8, 166.5, 165.7, 149.2, 142.2, 131.7, 131.4, 131.1, 128.8, 129.7, 129.3, 129.0, 128.9, 125.6, 124.8, 122.5, 118.9, 108.5, 67.5, 65.4, 53.7, 30.2, 26.7 ; ir (neat film nacl) 2096, 1718, 1637, 1533, 1475, 1358, 1232, 1184, 760 cm ; hrms (mm : esi - apci+) m / z calcd for c23h21n2o7 [m + h ] 437.1343, found 437.1298. an oven - dried flask was charged with ester 50 (1.3 g, 3.05 mmol, 1.00 equiv), sealed with a rubber stopper, and evacuated. the flask was brought in a glovebox, and pd(pph3)4 (84 mg, 75.0 mol, 0.025 equiv) was added. the flask was brought out of the drybox and thf (60 ml) was added. column chromatography using a teledyne isco combiflash rf (sio2, 80 g column, 25 50% etoac in hexanes) afforded the desired protected alkylation product 51 (1.1 g, 90% yield) as a colorless solid : rf = 0.40 (1:1 hexane / etoac) ; h nmr (400 mhz, cdcl3) 7.63 (dd, j = 7.9, 1.6 hz, 1h), 7.18 (dt, j = 7.8, 1.4 hz, 1h), 7.12 (dt, j = 7.5, 1.7 hz, 1h), 7.07 (dt, j = 7.7, 1.1 hz, 1h), 7.00 (d, j = 7.7 hz, 1h), 6.85 (dd, j = 8.0, 1.3 hz, 1h), 6.68 (d, j = 7.7 hz, 1h), 6.64 (dt, j = 7.7, 1.0 hz, 1h), 5.55 (tdd, j = 17.0, 10.2, 6.7 hz, 1h), 5.33 (ddd, j = 11.6, 10.0, 3.4 hz, 1h), 5.01 (ddd, j = 17.1, 3.0, 1.5 hz, 1h), 4.93 (ddd, j = 10.3, 2.7, 1.2 hz, 1h), 4.68 (td, j = 11.6, 4.7 hz, 1h), 3.45 (tdd, j = 15.6, 6.6, 1.3 hz, 1h), 3.26 (s, 3h), 2.99 (tdd, j = 9.0, 6.9, 1.3 hz, 1h), 2.87 (ddd, j = 14.6, 10.0, 4.6 hz, 1h), 2.21 (ddd, j = 14.7, 4.8, 3.5 hz, 1h) ; c nmr (100 mhz, cdcl3) 177.3, 168.5, 149.8, 142.1, 133.9, 133.1, 131.1, 130.6, 130.4, 128.6, 127.8, 125.7, 124.8, 122.2, 119.0, 107.6, 64.8, 54.2, 54.0, 43.7, 30.7, 26.5 ; ir (neat film nacl) 1701, 1614, 1531, 1473, 1356, 1300, 1259, 1202, 1105, 929, 739 cm ; hrms (mm : esi - apci+) m / z calcd for c22h21n2o5 [m + h ] 393.1445, found 393.1458. a solution of alkene 62 (47.1 mg, 100 mol, 1.00 equiv) in a mixture of ch2cl2 (2.5 ml) and meoh (2.5 ml) in a schlenk flask hooked up to an ozone generator was purged with oxygen gas at 78 c (5 min, flow 0.25). then the ozone generator was turned on (low medium setting), and an ozone / oxygen gas mixture was bubbled through the reaction. the progress of the reaction was checked via tlc (9:1 hexanes / ch2cl2) in short time intervals (12 min). upon completion of the reaction, the mixture was purged with oxygen gas for 5 min, and dms (36.0 g, 500 mol, 5.00 equiv) was added. the reaction mixture was slowly warmed to ambient temperature, and stirred for 16 h. the residue was purified by column chromatography (9:1 ch2cl2:etoac) on silica gel to afford aldehyde 63 (44 mg, 94% yield) : rf = 0.28 (9:1 ch2cl2/etoac) ; h nmr (500 mhz, dmso - d6) 8.64 (d, j = 3.4 hz, 1h), 7.837.79 (m, 1h), 7.657.59 (m, 1h), 7.437.39 (m, 1h), 7.31 (t, j = 8.0 hz, 1h), 7.21 (d, j = 7.9 hz, 1h), 7.05 (d, j = 8.1 hz, 1h), 6.92 (d, j = 8.3 hz, 1h), 5.15 (ddd, j = 12.5, 10.8, 4.7 hz, 1h), 4.59 (dd, j = 11.1, 6.9 hz, 1h), 3.94 (ddd, j = 14.5, 12.6, 7.1 hz, 1h), 3.22 (s, 3h), 3.08 (d, j = 17.0 hz, 1h), 2.67 (dd, j = 17.0, 3.5 hz, 1h), 1.96 (dd, j = 14.7, 4.6 hz, 1h) ; c nmr (125 mhz, dmso) 198.0, 174.8, 171.0, 151.5, 146.3, 133.3, 131.9, 131.3, 129.9, 129.7, 127.6, 125.5, 124.6, 122.4, 109.3, 65.4, 55.6, 52.6, 49.8, 26.4, 22.3 ; ir (neat film nacl) 1695, 1600, 1528, 1458, 1354, 1294, 1222, 1118, 850, 787 cm ; hrms (mm : esi - apci+) m / z calcd for c21h18brn2o6 [m + h ] 473.0343, found 473.0346. to a suspension of aldehyde 63 (47.3 mg, 100 mol, 1.00 equiv) and the acetic acid ammonium salt of p - methoxybenzylamine (59.2 mg, 300 mol, 3.00 equiv) in meoh (4 ml) was added nabh3cn (2.60 mg, 300 mol, 3.00 equiv) in thf (2 ml). the reaction mixture was stirred at ambient temperature for 16 h (conversion of the suspension to a clear, colorless solution usually indicated the completion of the reaction) and then concentrated under reduced pressure. column chromatography using a teledyne isco combiflash rf (sio2, 12 g column, 1. 1:1 1:4 hexanes / etoac) yielded lactam 65 (39.7 mg, 67% yield) as a colorless solid : rf = 0.12 (19:1 ch2cl2/meoh) ; h nmr (500 mhz, cdcl3) 9.10 (dd, j = 8.3, 1.4 hz, 1h), 7.597.55 (m, 1h), 7.43 (ddd, j = 8.4, 7.3, 1.3 hz, 1h), 7.36 (dd, j = 8.0, 1.6 hz, 1h), 7.17 (t, j = 7.9 hz, 1h), 7.10 (dd, j = 8.2, 1.0 hz, 1h), 7.067.02 (m, 2h), 6.816.76 (m, 3h), 4.71 (d, j = 14.6 hz, 1h), 3.98 (d, j = 14.6 hz, 1h), 3.76 (s, 3h), 3.533.49 (m, 1h), 3.18 (s, 3h), 3.163.08 (m, 3h), 2.90 (ddd, j = 9.6, 8.5, 3.2 hz, 1h), 2.78 (dt, j = 9.6, 7.3 hz, 1h), 2.25 (ddd, j = 14.1, 7.1, 3.1 hz, 1h), 2.14 (ddd, j = 14.0, 8.6, 7.6 hz, 1h) ; c nmr (125 mhz, cdcl3) 177.7, 171.7, 159.2, 152.6, 147.2, 134.4, 130.9, 130.4, 130.3, 129.9, 129.0, 128.0, 127.9, 126.3, 125.5, 122.4, 114.1, 107.3, 60.1, 58.5, 55.7, 55.4, 47.4, 44.0, 32.1, 27.5, 26.8 ; ir (neat film nacl) 3459, 2931, 1682, 1601, 1574, 1530, 1457, 1360, 1249, 1176, 1037, 910, 849, 783, 731 cm ; hrms (mm : esi - apci+) m / z calcd for c29h29brn3o6 [m + h ] 594.1234, found 594.1230. to a solution of aldehyde 69 (100 mg, 0.15 mmol, 1.00 equiv) and (p - methoxybenzyl)ammonium acetate (90 mg, 0.46 mmol, 3.00 equiv) in methanol (7.6 ml) was added nabh3cn (21 mg, 0.30 mmol, 2.00 equiv) in thf (3.8 ml) at 0 c. the reaction mixture was slowly warmed to 23 c and stirred overnight. the reaction mixture was washed with etoac (3 10 ml), and brine. the residue was purified by column chromatography (4:1 hexanes / etoac) on silica gel to afford lactam 54 (112 mg, 95% yield) : rf = 0.20 (4:1 hexanes / etoac) ; h nmr (500 mhz, cdcl3) 12.14 (s, 1h), 8.00 (d, j = 8.3 hz, 1h), 7.18 (d, j = 8.3 hz, 2h), 7.157.10 (m, 2h), 7.05 (q, j = 8.5, 7.9 hz, 2h), 6.88 (t, j = 7.7 hz, 1h), 6.856.80 (m, 2h), 6.49 (d, j = 7.7 hz, 1h), 4.80 (d, j = 14.4 hz, 1h), 4.36 (d, j = 14.4 hz, 1h), 3.78 (s, 3h), 3.70 (s, 1h), 3.63 (s, 3h), 3.52 (td, j = 6.9, 3.6 hz, 1h), 3.433.36 (m, 1h), 3.27 (td, j = 9.3, 5.7 hz, 1h), 3.17 (t, j = 9.6 hz, 1h), 2.87 (ddd, j = 13.3, 6.1, 3.5 hz, 1h), 2.662.60 (m, 1h), 2.56 (s, 3h), 2.542.49 (m, 1h), 0.85 (q, j = 3.8 hz, 21h) ; c nmr (125 mhz, cdcl3) 175.94, 173.92, 159.34, 154.32, 146.89, 139.65, 132.30, 130.98, 130.07, 129.75, 128.88, 128.50, 127.57, 127.42, 126.71, 126.45, 121.74, 120.99, 120.75, 114.18, 107.15, 68.21, 61.31, 60.86, 60.43, 55.34, 51.42, 47.61, 44.98, 31.20, 25.78, 17.86, 11.88 ; ir (neat film nacl) 2944, 2865, 2073, 1716, 1667, 1604, 1513, 1455, 1247, 1227, 1109, 1069, 1034, 883, 761 cm ; hrms (mm : esi - apci+) m / z calcd for c40h53brn3o6si [m + h ] 778.2882, found 778.2879. to a solution of amide 54 (0.32 g, 0.41 mmol, 1.00 equiv) in thf (41.1 ml) was added alh3me2net (0.5 m in toluene ; 1.64 ml, 0.82 mmol, 2.00 equiv) dropwise at 0 c. the reaction solution was concentrated in vacuo and purified by column chromatography (4:1 hexanes / etoac) to afford aminal 71 (0.19 g, 61% yield) : rf = 0.20 (4:1 hexanes / etoac) ; h nmr (300 mhz, cdcl3) 7.587.47 (m, 1h), 7.21 (d, j = 8.2 hz, 2h), 7.046.93 (m, 3h), 6.80 (dq, j = 14.8, 7.4 hz, 4h), 6.396.24 (m, 2h), 4.30 (m, 1h), 3.923.79 (m, 4h), 3.78 (s, 3h), 3.68 (dd, j = 10.2, 5.0 hz, 1h), 3.573.43 (m, 1h), 3.03 (s, 3h), 2.65 (m, 4h), 2.26 (dt, j = 15.0, 7.2 hz, 1h), 2.09 (dd, j = 11.9, 4.6 hz, 1h), 0.87 (d, j = 3.2 hz, 21h) ; c nmr (125 mhz, cdcl3) 176.0, 175.7, 160.6, 146.3, 141.7, 133.9, 132.2, 130.3, 130.1, 126.8, 123.6, 123.2, 123.1, 115.1, 114.6, 113.9, 107.3, 107.1, 106.6, 82.4, 60.7, 60.1, 57.2, 55.3, 52.6, 51.0, 31.9, 31.1, 30.7, 29.7, 26.1, 17.7, 11.8 ; ir (neat film nacl) 2943, 1722, 1604, 1464, 1386, 1344, 1254, 1107, 1033, 885, 760 cm ; hrms (mm : esi - apci+) m / z calcd for c40h53brn3o5si [m + h ] 762.2932, found 762.2936. to a solution of silyl ether 71 (98 mg, 0.13 mmol, 1.00 equiv) in thf (1.28 ml) was added tbaf (0.15 ml, 1.0 m solution in thf) at 0 c. the reaction mixture was stirred for 3 h at 23 c and quenched with satd nh4cl. the reaction mixture was washed with etoac (3 1.5 ml) and brine. the residue was purified by column chromatography (2:1 hexanes / etoac) on silica gel to afford pmb - protected aminal compound (78 mg, 98% yield). to a solution of pmb - protected aminal compound (96 mg, 0.16 mmol, 1.00 equiv) in ch2cl2 (3.1 ml) and h2o (0.8 ml) was added ddq (53 mg, 0.23 mmol, 1.50 equiv) in portions over 30 min at 0 c. the reaction mixture was washed with ch2cl2 (3 2.5 ml) and brine. the residue was purified by column chromatography (4:1 ch2cl2/acetone) on silica gel to afford aminal 72 (64 mg, 85% yield) : rf = 0.31 (4:1 ch2cl2/acetone) ; h nmr (600 mhz, cdcl3) 7.50 (d, j = 8.1 hz, 1h), 7.016.96 (m, 2h), 6.84 (p, j = 8.6 hz, 1h), 6.78 (d, j = 7.6 hz, 1h), 6.73 (t, j = 7.5 hz, 1h), 6.48 (s, 1h), 6.35 (d, j = 7.8 hz, 1h), 3.87 (d, j = 17.2 hz, 3h), 3.66 (ddd, j = 10.8, 6.8, 3.7 hz, 1h), 3.35 (dt, j = 10.9, 5.3 hz, 1h), 3.11 (s, 3h), 3.01 (dd, j = 10.0, 6.5 hz, 1h), 2.802.76 (m, 1h), 2.762.62 (m, 2h), 2.50 (td, j = 10.3, 4.7 hz, 1h), 2.24 (br, s, 1h), 2.20 (dd, j = 12.7, 4.8 hz, 1h) ; c nmr (125 mhz, cdcl3) 177.6, 152.8, 146.1, 142.7, 130.4, 129.7, 128.9, 127.4, 127.2, 122.8, 121.6, 118.5, 113.8, 106.8, 80.5, 63.4, 60.0, 57.2, 52.6, 44.6, 35.1, 31.5, 26.3 ; ir (neat film nacl) 3417, 2925, 1710, 1604, 1487, 1458, 1392, 1362, 1272, 1093, 756 cm ; hrms (mm : esi - apci+) m / z calcd for c23h25brn3o4 [m + h ] 486.1023, found 486.1014. to a solution of lactam 54 (10.6 mg, 0.0136 mmol, 1.00 equiv) in thf (1.36 ml) was added lialh4 (5.2 mg, 0.136 mmol, 10.0 equiv) in portions at 0 c. the reaction mixture was washed with etoac (3 2 ml) and brine. the residue was purified by column chromatography (4:1 hexanes / etoac) to afford oxindole 73 (7.9 mg, 83% yield) : rf = 0.25 (4:1 hexanes / etoac) ; h nmr (600 mhz, cdcl3) 9.64 (br, s, 1h), 8.02 (d, j = 7.5 hz, 1h), 7.60 (br, s, 1h), 7.13 (d, j = 8.4 hz, 3h), 7.09 (t, j = 7.6 hz, 1h), 7.04 (t, j = 7.7 hz, 1h), 6.97 (s, 1h), 6.83 (d, j = 8.3 hz, 3h), 6.38 (d, j = 7.7 hz, 1h), 4.55 (d, j = 14.5 hz, 1h), 4.44 (d, j = 14.5 hz, 1h), 3.77 (s, 3h), 3.71 (s, 3h), 3.313.27 (m, 1h), 3.24 (br, s, 2h), 3.14 (br, s, 1h), 3.053.00 (m, 1h), 2.85 (s, 3h), 2.72 (br, s, 1h), 2.52 (m, 1h), 2.41 (br, s, 1h), 0.86 (q, j = 7.5, 6.2 hz, 21h) ; c nmr (125 mhz, cdcl3) 177.5, 159.4, 154.7, 143.7, 129.6, 129.4, 129.3, 129.2, 128.6, 128.4, 127.7, 127.5, 127.4, 122.2, 122.0, 121.8, 114.3, 107.3, 60.0, 56.6, 55.4, 52.1, 47.1, 44.0, 34.3, 29.9, 26.0, 18.0, 12.0 ; ir (neat film nacl) 2941, 1732, 1711, 1610, 1515, 1442, 1375, 1248, 1225, 1105, 1070, 1036, 750 cm ; hrms (mm : esi - apci+) m / z calcd for c40h54n3o6si [m + h ] 700.3776, found 700.3776. to a solution of lactam 54 (70 mg, 0.090 mmol, 1.00 equiv) in ch2cl2 (9 ml) was added tf2o (45 ml, 0.27 mmol, 3.00 equiv) dropwise at 0 c. the reaction mixture was slowly warmed to 23 c and stirred for 2 h. the solution was neutralized by adding satd nahco3. the reaction mixture was washed with ch2cl2 (3 5 ml) and brine. the residue was purified by column chromatography (2:1 hexanes / etoac) on silica gel to afford propellane hexacycle 76 (52 mg, 95% yield) : rf = 0.25 (3:1 hexanes / etoac) ; h nmr (500 mhz, cdcl3) 7.67 (s, 1h), 7.167.11 (m, 3h), 6.956.93 (m, 2h), 6.78 (d, j = 8.6 hz, 2h), 6.73 (td, j = 7.6, 1.1 hz, 1h), 6.60 (t, j = 4.4 hz, 1h), 6.36 (dd, j = 7.6, 1.5 hz, 1h), 4.53 (dt, j = 11.5, 5.8 hz, 1h), 4.47 (d, j = 14.9 hz, 1h), 4.234.18 (m, 1h), 3.87 (s, 3h), 3.76 (s, 3h), 3.72 (d, j = 13.7 hz, 1h), 3.21 (s, 3h), 3.01 (ddd, j = 14.4, 11.7, 8.4 hz, 1h), 2.772.73 (m, 1h), 2.54 (q, j = 9.3 hz, 1h), 2.13 (td, j = 8.8, 5.3 hz, 1h), 1.93 (dd, j = 14.2, 6.3 hz, 1h), 1.801.75 (m, 1h) ; c nmr (125 mhz, cdcl3) 178.0, 158.5, 153.7, 145.8, 142.5, 132.0, 130.9, 130.1, 129.5, 129.4, 128.9, 128.1, 124.6, 122.9, 121.5, 116.1, 113.6, 112.1, 106.6, 58.2, 57.0, 55.4, 54.4, 52.7, 50.2, 47.9, 33.7, 26.6, 23.2 ; ir (neat film nacl) 1718, 1601, 1575, 1513, 1484, 1455, 1365, 1245, 1134, 1099, 1037, 912, 764, 731 cm ; hrms (mm : esi - apci+) m / z calcd for c31h31brn3o5 [m + h ] 604.1442, found 604.1433. to a solution of oxindole 76 (46 mg, 0.075 mmol, 1.00 equiv) in ch2cl2 (3.8 ml) and h2o (0.94 ml) was added ddq (34 mg, 0.15 mmol, 2.00 equiv) at 0 c. the reaction mixture was slowly warmed to 23 c and stirred for 2 h. the solution was quenched with satd nahco3. the reaction mixture was washed with ch2cl2 (3 3.0 ml) and brine. the residue was purified by column chromatography (4:1 ch2cl2/acetone) on silica gel to afford propellane hexacycle 77 (33 mg, 92% yield) : rf = 0.1 (1:1 hexane / etoac) ; h nmr (500 mhz, cdcl3) 7.70 (d, j = 8.2 hz, 1h), 7.16 (ddd, j = 8.3, 7.4, 1.4 hz, 1h), 7.03 (dd, j = 8.2, 7.6 hz, 1h), 6.95 (dd, j = 8.2, 1.1 hz, 1h), 6.72 (ddd, j = 14.7, 7.6, 1.1 hz, 2h), 6.26 (ddd, j = 7.6, 1.4, 0.5 hz, 1h), 4.47 (td, j = 11.2, 6.6 hz, 1h), 4.124.07 (m, 1h), 3.93 (s, 3h), 3.23 (s, 3h), 3.14 (dd, j = 9.4, 6.0 hz, 1h), 3.00 (ddd, j = 14.4, 11.2, 8.2 hz, 1h), 2.682.57 (m, 2h), 1.891.85 (m, 1h), 1.82 (ddd, j = 14.4, 6.6, 1.6 hz, 1h) ; c nmr (125 mhz, cdcl3) 177.9, 153.6, 145.8, 142.4, 133.3, 130.4, 129.7, 129.2, 128.1, 125.7, 123.2, 122.5, 114.3, 111.6, 106.9, 58.5, 55.7, 53.8, 53.0, 43.3, 36.5, 26.6, 23.5 ; ir (neat film nacl) 2958, 1713, 1602, 1485, 1446, 1373, 1242, 1095, 754 cm ; hrms (mm : esi - apci+) m / z calcd for c23h23brn3o4 [m + h ] 484.0866, found 484.0874. to a solution of propellane hexacycle 76 (13 mg, 0.021 mmol, 1.00 equiv) in ch2cl2 (2.14 ml) was added dibal (1.0 m in thf ; 0.11 ml, 0.11 mmol, 5.00 equiv) dropwise at 78 c. dibal (1.0 m in thf ; 21.4 ml, 21.4 mmol, 1.00 equiv) was added dropwise at 0 c and the mixture stirred for 1 h. then, dibal (1.0 m in thf ; 21.4 ml, 21.4 mmol, 1.00 equiv) was added one more time dropwise at 0 c and the mixture stirred for another 1 h. the reaction mixture was warmed to 23 c, and et2alcl (1.0 m in hexane ; 42.8 ml, 42.8 mmol, 2.00 equiv) was added dropwise. the mixture was stirred for 30 min and quenched with satd nh4cl and satd potassium sodium tartrate. the reaction mixture was washed with ch2cl2 (3 2.0 ml) and brine. the residue was purified by column chromatography (1:1 hexane / etoac) on silica gel to afford aminal 81 (10.5 mg, 87% yield) : rf = 0.25 (1:1 hexane / etoac) ; h nmr (500 mhz, cdcl3) 7.39 (d, j = 7.7 hz, 1h), 7.247.22 (m, 2h), 7.17 (m, 1h), 7.087.03 (m, 1h), 6.876.84 (m, 2h), 6.82 (t, j = 7.9 hz, 1h), 6.71 (dd, j = 8.0, 1.0 hz, 1h), 6.23 (br, s, 1h), 6.01 (dd, j = 7.9, 1.0 hz, 1h), 5.15 (d, j = 14.5 hz, 1h), 3.94 (d, j = 14.5 hz, 1h), 3.84 (s, 3h), 3.79 (s, 3h), 3.723.63 (m, 2h), 3.40 (dd, j = 9.5, 7.7 hz, 1h), 3.12 (td, j = 9.7, 1.5 hz, 1h), 3.103.04 (m, 1h), 2.892.82 (m, 1h), 2.48 (s, 3h), 2.32 (dt, j = 14.2, 7.2 hz, 1h), 1.65 (dt, j = 14.7, 9.7 hz, 1h), 1.59 (br, s, 1h) ; c nmr (125 mhz, cdcl3) 170.5, 159.2, 152.7, 139.2, 136.3, 134.0, 130.4, 129.8, 128.8, 126.8, 126.7, 126.1, 125.2, 125.0, 122.9, 122.4, 114.2, 104.7, 83.3, 61.0, 60.5, 55.4, 53.6, 53.4, 47.1, 44.3, 35.3, 33.0, 31.1 ; ir (neat film nacl) 2922, 1689, 1597, 1512, 1444, 1334, 1249, 1178, 1032, 754 cm ; hrms (mm : esi - apci+) m / z calcd for c31h33brn3o5 [m + h ] 606.1598, found 606.1592. to a solution of amide 89 (20 mg, 0.0239 mmol, 1.00 equiv) in ch2cl2 (2.39 ml), was added tf2o (0.0121 ml, 0.0718 mmol, 3.00 equiv) dropwise at 0 c. the reaction mixture was slowly warmed to 23 c and the mixture stirred for 2 h. after the reaction was complete, the solution was brought to ph 10.511.0 by addition of satd nahco3. the reaction mixture was extracted with etoac (3 3 ml) and washed with brine. the residue was purified by column chromatography (2:1 hexanes / etoac) on silica gel to afford tetrahydroazepine 91 (9.4 mg, 70% yield) : rf = 0.33 (2:1 hexanes / etoac) ; h nmr (600 mhz, cdcl3) 8.04 (d, j = 8.1 hz, 1h), 7.69 (t, j = 7.6 hz, 1h), 7.53 (d, j = 7.8 hz, 1h), 7.43 (t, j = 7.8 hz, 1h), 7.19 (t, j = 7.7 hz, 1h), 7.09 (d, j = 2.1 hz, 1h), 6.98 (dd, j = 8.3, 2.0 hz, 1h), 6.82 (d, j = 7.8 hz, 1h), 6.73 (t, j = 7.7 hz, 1h), 6.70 (dd, j = 8.4, 1.3 hz, 1h), 6.18 (d, j = 7.6 hz, 1h), 5.08 (d, j = 17.0 hz, 1h), 4.47 (d, j = 17.0 hz, 1h), 3.89 (br, s, 1h), 3.78 (q, j = 13.0, 12.0 hz, 1h), 3.313.26 (m, 2h), 3.23 (td, j = 9.3, 8.5, 4.1 hz, 1h), 3.18 (s, 3h), 2.962.89 (m, 2h), 2.70 (dt, j = 13.5, 8.5 hz, 1h), 1.42 (d, j = 13.3 hz, 1h) ; c nmr (125 mhz, cdcl3) 180.4, 175.1, 150.3, 148.3, 143.8, 134.2, 132.5, 131.1, 131.1, 129.4, 129.2, 128.3, 127.7, 126.0, 125.3, 125.0, 124.6, 121.7, 121.3, 108.0, 58.8, 50.3, 44.6, 44.1, 43.4, 36.1, 27.6, 26.4 ; ir (neat film nacl) 3343, 2942, 1703, 1611, 1588, 1524, 1471, 1357, 1285, 1137, 1106, 1065, 984, 858, 732 cm ; hrms (mm : esi - apci+) m / z calcd for c28h26brn4o4 [m + h ] 561.1132, found 561.1165. | expedient synthetic approaches to the highly functionalized polycyclic alkaloids communesin f and perophoramidine are described using a unified approach featuring a key decarboxylative allylic alkylation to access a crucial and highly congested 3,3-disubstituted oxindole. described are two distinct, stereoselective alkylations that produce structures in divergent diastereomeric series possessing the critical vicinal all - carbon quaternary centers needed for each synthesis. synthetic studies toward these challenging core structures have revealed a number of unanticipated modes of reactivity inherent to these complex alkaloid scaffolds. additionally, several novel and interesting intermediates en route to the target natural products, such as an intriguing propellane hexacyclic oxindole encountered in the communesin f sequence, are disclosed. indeed, such unanticipated structures may prove to be convenient strategic intermediates in future syntheses. |
despite various developments in the field of cancer diagnosis and treatment, many patients develop incurable disease. these patients, though incurable, should be offered appropriate treatment to maximize their quality of life. palliative care, as defined by world health organization (who), is the active total care of patient whose disease is not responsive to curative treatment. a patient with advanced incurable disease may tolerate the anti - neoplastic treatment poorly and may become further disabled. thus, an oncologist should try to maintain an intricate balance between expected symptom relief and the possible toxicities of the treatment. palliative radiotherapy (prt) is required in 30 - 50% of all cancer patients, and the primary aim of prt is to provide adequate pain and symptom relief. there might be complex issues pertaining to pain, physical symptoms, and psychosocial aspects coexisting during prt, which needs to be adequately addressed by a palliative care unit. besides providing pain relief, prt is useful to prevent / treat collapse or fracture in case of bone metastasis, to reduce headache and edema in patients with cranial metastasis, and to control distressing symptoms of rapid primary growth, e.g. prt in cases of recurrent breast cancer. in this article, we present a review of the medical records of the patients who received prt in our institute. such a review is required for the characterization of the requirements of the patients and the formulation of institutional policies. in total, 516 patients, who received prt at our institute from january 2012 to december 2012 and whose complete records were available for analysis, were selected for this retrospective study. our institute is a regional cancer center with separate palliative care facilities, thus providing comprehensive palliative management of patients with advanced incurable disease. the treatment goal in such patients is to achieve the best possible quality of life. patients who underwent prt were included in this study, irrespective of age, sex, primary disease, site of radiotherapy, or site of metastasis, the only selection criteria being availability of sufficient treatment details and records of sociodemographic parameters. the following data were retrieved from the medical records and radiotherapy files : age, sex, symptoms, site of primary, site of metastasis (if any), response to prt, and last follow up. the descriptive statistics of the prt data were evaluated in terms of frequencies and percentages to allow comparisons. the prt dose ranged from 8 gy to 30 gy given in the fractionation of 3 - 8 gy per fraction. for bone metastasis, two fractionation schedules were primarily used : 8 gy in single fraction or 30 gy in 10 fractions. the latter was also used for cranial metastasis, superior vena caval (svc) syndrome, and achieving hemostasis. the allocation to the fractionation schedule was based on the discretion of radiation oncologist and based on the patient 's general condition. lead fiducial markers were used to obtain portal film and confirm the dose delivery. the response to cranial radiotherapy was assessed by improvement in performance status and resolution of symptoms, such as headache and vomiting. the response to prt for svc was assessed by relief from dyspnea and resolution of symptoms of vein engorgement and pain. all responses were recorded as one of the four categories : complete relief, partial relief, stable, and progressive status. the second line prt schedule for patients with bone metastasis used was 600 cgy in single fraction in all the patients in whom pain or weakness of the lower limbs reappeared. this second line prt was used after a minimum interval of 4 weeks. in patients with advanced gynecologic malignancies, two schedules were used : 5 fractions of 300 cgy or 3 fractions of 400 cgy. the fractionation used for patients with advanced bladder carcinoma was 300 cgy five fractions followed by conventional fractionation in patients with better life expectancy. in patients with fungating lesions and large neck nodes, two prt schedules have been used in our institute with a fractionation of 600 cgy given weekly for 5 weeks and 300 cgy 14 fractions given 5 days a week. in patients with fungating lesions and large neck nodes due to head and neck cancer, two prt schedules have been used in our institute with a fractionation of 600 cgy given weekly for 5 weeks and 300 cgy 14 fractions given five days a week. conventional fractionation was employed to complete the curative dose in those achieving good response and good life expectancy. out of 516 patients analyzed in this study, 73% patients were male ; the median age of the patients receiving prt was 62 years (range 13 - 83). the primary site of the disease was lung in 38.7% of the patients followed by head and neck (19.4%) and breast (10.8%). patients with gynecologic malignancies and prostate cancer accounted for 21.8% and 6.1% of the cases, respectively. about 59% patients were stage iv at the time of prescription of prt while 38% belonged to stage iii. distant metastasis was identified in 52% of the patients, with the most common site being bones (64%) followed by brain (26%) [table 1 ]. about 26% of the patients had received chemotherapy before starting prt while concurrent chemoradiotherapy was given to 8% of the patients. the median dose prescribed was 30 gy (range 8 - 36 gy) delivered in 1 - 12 fractions over the duration of 1 - 18 days. upfront prt was prescribed to 64% of the patients due to metastatic presentation or poor performance status of the patients. the most common indication of prt was pain (56.8% of the cases), followed by cytostatic prt (19.8%) and raised ict (12.4%) [table 2 ]. cytostatic prt was aimed to control bleeding, to relieve pressure symptoms, and to control excess discharge or pain arising from ulceration. patient characteristics treatment characteristics about 6% patients had complete relief for all symptoms for which prt was indicated, while 37% patients got partial relief ; thus, the overall response was about 43%. about 5% patients died within 2 weeks of completion of prt while 11% were lost to follow up [table 3 ]. the median follow - up of the patients receiving prt was 22 weeks (range 9 - 256 days). table 4 depicts the median and range of palliative radiotherapy dose, fractions and follow up duration. the median duration of response of bone metastasis to prt was about 36 days after completion of prt. the overall response rate was 54% ; the retreatment rate was 16%. in patients who received palliative cranial radiotherapy, the overall median survival was 2.6 months and survival at one year was 8.57%. in patients receiving prt for svc syndrome, the response to prt for head and neck cancer was about 46% with 20% patients proceeding to receive the curative dose. out of 516 patients analyzed in this study, 73% patients were male ; the median age of the patients receiving prt was 62 years (range 13 - 83). the primary site of the disease was lung in 38.7% of the patients followed by head and neck (19.4%) and breast (10.8%). patients with gynecologic malignancies and prostate cancer accounted for 21.8% and 6.1% of the cases, respectively. about 59% patients were stage iv at the time of prescription of prt while 38% belonged to stage iii. distant metastasis was identified in 52% of the patients, with the most common site being bones (64%) followed by brain (26%) [table 1 ]. about 26% of the patients had received chemotherapy before starting prt while concurrent chemoradiotherapy was given to 8% of the patients. the median dose prescribed was 30 gy (range 8 - 36 gy) delivered in 1 - 12 fractions over the duration of 1 - 18 days. upfront prt was prescribed to 64% of the patients due to metastatic presentation or poor performance status of the patients. the most common indication of prt was pain (56.8% of the cases), followed by cytostatic prt (19.8%) and raised ict (12.4%) [table 2 ]. cytostatic prt was aimed to control bleeding, to relieve pressure symptoms, and to control excess discharge or pain arising from ulceration. about 6% patients had complete relief for all symptoms for which prt was indicated, while 37% patients got partial relief ; thus, the overall response was about 43%. about 5% patients died within 2 weeks of completion of prt while 11% were lost to follow up [table 3 ]. the median follow - up of the patients receiving prt was 22 weeks (range 9 - 256 days). table 4 depicts the median and range of palliative radiotherapy dose, fractions and follow up duration. the median duration of response of bone metastasis to prt was about 36 days after completion of prt. the overall response rate was 54% ; the retreatment rate was 16%. in patients who received palliative cranial radiotherapy, the overall median survival was 2.6 months and survival at one year was 8.57%. in patients receiving prt for svc syndrome, the response to prt for head and neck cancer was about 46% with 20% patients proceeding to receive the curative dose. palliative treatment is aimed to provide symptomatic relief and the best achievable quality of life in patients suffering from incurable diseases. there is an important misconception among the physicians who still refer to palliative medicine to be a branch completely focused on the care of terminally ill patients. thus, there is an urgent need to spread awareness of the importance of palliative medicine. prt is an important tool for an oncologist to provide effective pain relief, to reduce the intracranial tension, to relieve compressive symptoms and dyspnea, to enable regression of fungating mass, and to achieve hemostasis in the appropriate settings. most of the schedules used for prt are hypofractionated based on the shorter life expectancy and problems of protracted treatment with conventional fractionation. the standard treatment for palliation of bone metastases has been daily treatment for 2 - 3 weeks with doses of 30 - 35 gy in 10 - 14 treatments. in our patients with skeletal metastasis, the fractionation schedule mostly used were 8 gy in single fraction or 30 gy in 10 fractions depending upon the patients general condition and choice of the consultant. this is in accordance with the result of multiple randomized, prospective trials in the last 30 years, which have concluded that single - dose treatment of 8 gy provides pain relief similar to longer treatment regimens. in most of these studies, there were no differences reported in pain relief or pain medication requirements between the treatment regimens. the overall response rate in our study was 54% while the retreatment rate was 21% after single fraction and 4% after multiple fractions. this is in accordance with previous studies in which the need for retreatment ranged from 11% to 29% after single - fraction therapy, compared with 0 - 24% after multiple - fraction treatment. whole brain radiotherapy (wbrt) is an effective measure to control symptoms of raised intracranial tension in patients with multiple cranial metastases. wbrt was delivered in 24% patients, with majority of patients having primary of lung (54%) and breast (28%). the most common fractionation schedule used for cranial radiotherapy in our study patients was 300 cgy in 10 fractions delivered over two weeks. mehta. reported it to improve edema and neurological deficits in about 70% of the patients. in our patients, injectable dexamethasone was started by admitting the patients immediately after diagnosis of brain metastasis along with an injection of mannitol. reported that the median survival could be increased to approximately 4 - 6 months in such patients by addition of wbrt. this may be ascribed to delayed presentation of our patients with multiple cranial metastases with poor overall general condition. thoracic symptoms including svc syndrome are effectively palliated with hypofractionated radiotherapy. in our study, 6.9% patients had features of svc syndrome. other thoracic symptoms for where prt was used included dyspnea, chest pain, and hemoptysis. various studies have reported employing either 16 gy or 17 gy in 2 fractions or 20 gy in 5 fractions for prt of non - small cell lung cancer. few severe side effects were noted, and symptom response was consistent between the studies. in patients with acute, life - threatening symptoms, relief of symptoms (dyspnea and venous engorgement) was obtained in median 6 days in about 73% of the patients. locally advanced pelvic malignancies may cause distressing symptoms, including pain, discharge, bleeding, and hydronephrosis leading to obstructive uropathy. in a phase ii rtog trial, patients with advanced gynecologic and genitourinary malignancies were treated with a total dose of 44.4 gy in 12 fractions. the dose was given as 3.7-gy fractions twice daily for 2 days with 3 - 6-week breaks after 14.8 gy and 29.6 gy. the patients had acceptable acute and long - term side - effect rates, with an overall response rate of about 40%. in our patients, two schedules were used : 5 fractions of 300 cgy or 3 fractions of 400 cgy. hydronephrosis was relieved in 54% patients, and they were given conventional fractionation to complete the dose. in patients of locally advanced prostate cancer presenting with lower urinary tract symptoms or hematuria, prt led to response in 78% of the patients., in which they observed a response of 88% by employing 35 gy in 15 fractions or 24 gy in 3 weekly fractions. in a randomized trial by duchesne., similar palliation rate of about 70% along with similar side effects and survival rates were obtained by using either 35 gy in 10 fractions or 21 gy in 3 fractions for patients with locally advanced, inoperable bladder carcinoma. the fractionation used in our study was 300 cgy five fractions followed by conventional fractionation in patients with good life expectancy. our patients had 84% response rate in terms of resolution of hematuria. in developing countries, patients of head and neck cancer usually present with locoregionally advanced disease. in patients with fungating lesions and large neck nodes, two prt schedules have been used in our institute with a fractionation of 600 cgy given weekly for 5 weeks and 300 cgy 14 fractions given five days a week. conventional fractionation is employed to complete the curative dose in those achieving good response and good life expectancy. the response to both the schedules is about 46% with no major differences in acute toxicities. the median survival was 7.6 months (2.5 - 11 months). in a study by mohanti. employing 20 gy in 5 fractions, symptom relief for pain, dysphagia, hoarseness, cough, and otalgia entitled the quad shot, consisting of 14 gy in 4 fractions given twice per day for 2 consecutive days, the median survival was 5.7 months. the regimen was repeated at 4-week intervals for an additional 2 courses if there was no tumor progression. many patients who receive treatment near the end of life require an intense effort to achieve transportation out of the home ; thus, the optimal intervention for this group is 1 visit that includes consultation, dose planning, and delivery of a single - fraction treatment a series of tasks that may be completed within 2 hours at most radiotherapy centers. the toxicity profile of radiotherapy has a clear advantage over systemic therapy as the deleterious effects are usually limited to the anatomic site of treatment and for a shorter time, whereas systemic therapy often cause functional deficits in several organ systems. the precise information about the quality of life was not available in the records ; hence, it could not be quoted in the study. however, there was qualitative improvement of the patients as mentioned in the data was included in the study. in addition, there was limited use of standard pain assessment scales in the records making the comparisons difficult. the standard treatment for palliation of bone metastases has been daily treatment for 2 - 3 weeks with doses of 30 - 35 gy in 10 - 14 treatments. in our patients with skeletal metastasis, the fractionation schedule mostly used were 8 gy in single fraction or 30 gy in 10 fractions depending upon the patients general condition and choice of the consultant. this is in accordance with the result of multiple randomized, prospective trials in the last 30 years, which have concluded that single - dose treatment of 8 gy provides pain relief similar to longer treatment regimens. in most of these studies, there were no differences reported in pain relief or pain medication requirements between the treatment regimens. the overall response rate in our study was 54% while the retreatment rate was 21% after single fraction and 4% after multiple fractions. this is in accordance with previous studies in which the need for retreatment ranged from 11% to 29% after single - fraction therapy, compared with 0 - 24% after multiple - fraction treatment. whole brain radiotherapy (wbrt) is an effective measure to control symptoms of raised intracranial tension in patients with multiple cranial metastases. wbrt was delivered in 24% patients, with majority of patients having primary of lung (54%) and breast (28%). the most common fractionation schedule used for cranial radiotherapy in our study patients was 300 cgy in 10 fractions delivered over two weeks. mehta. reported it to improve edema and neurological deficits in about 70% of the patients. in our patients, injectable dexamethasone was started by admitting the patients immediately after diagnosis of brain metastasis along with an injection of mannitol. reported that the median survival could be increased to approximately 4 - 6 months in such patients by addition of wbrt. this may be ascribed to delayed presentation of our patients with multiple cranial metastases with poor overall general condition. thoracic symptoms including svc syndrome are effectively palliated with hypofractionated radiotherapy. in our study, 6.9% patients had features of svc syndrome. other thoracic symptoms for where prt was used included dyspnea, chest pain, and hemoptysis. various studies have reported employing either 16 gy or 17 gy in 2 fractions or 20 gy in 5 fractions for prt of non - small cell lung cancer. few severe side effects were noted, and symptom response was consistent between the studies. in patients with acute, life - threatening symptoms, relief of symptoms (dyspnea and venous engorgement) was obtained in median 6 days in about 73% of the patients. knopp. reported that 85 - 90% of the patients attained symptomatic relief within three weeks. locally advanced pelvic malignancies may cause distressing symptoms, including pain, discharge, bleeding, and hydronephrosis leading to obstructive uropathy. in a phase ii rtog trial, patients with advanced gynecologic and genitourinary malignancies were treated with a total dose of 44.4 gy in 12 fractions. the dose was given as 3.7-gy fractions twice daily for 2 days with 3 - 6-week breaks after 14.8 gy and 29.6 gy. the patients had acceptable acute and long - term side - effect rates, with an overall response rate of about 40%. in our patients, two schedules were used : 5 fractions of 300 cgy or 3 fractions of 400 cgy. hydronephrosis was relieved in 54% patients, and they were given conventional fractionation to complete the dose. in patients of locally advanced prostate cancer presenting with lower urinary tract symptoms or hematuria, prt led to response in 78% of the patients., in which they observed a response of 88% by employing 35 gy in 15 fractions or 24 gy in 3 weekly fractions. in a randomized trial by duchesne., similar palliation rate of about 70% along with similar side effects and survival rates were obtained by using either 35 gy in 10 fractions or 21 gy in 3 fractions for patients with locally advanced, inoperable bladder carcinoma. the fractionation used in our study was 300 cgy five fractions followed by conventional fractionation in patients with good life expectancy. in developing countries, patients of head and neck cancer usually present with locoregionally advanced disease. in patients with fungating lesions and large neck nodes, two prt schedules have been used in our institute with a fractionation of 600 cgy given weekly for 5 weeks and 300 cgy 14 fractions given five days a week. conventional fractionation is employed to complete the curative dose in those achieving good response and good life expectancy. the response to both the schedules is about 46% with no major differences in acute toxicities. the median survival was 7.6 months (2.5 - 11 months). in a study by mohanti. employing 20 gy in 5 fractions, symptom relief for pain, dysphagia, hoarseness, cough, and otalgia was obtained in 47 - 59% of the patients following prt. in a regimen entitled the quad shot, consisting of 14 gy in 4 fractions given twice per day for 2 consecutive days, the median survival was 5.7 months. the regimen was repeated at 4-week intervals for an additional 2 courses if there was no tumor progression. many patients who receive treatment near the end of life require an intense effort to achieve transportation out of the home ; thus, the optimal intervention for this group is 1 visit that includes consultation, dose planning, and delivery of a single - fraction treatment a series of tasks that may be completed within 2 hours at most radiotherapy centers. the toxicity profile of radiotherapy has a clear advantage over systemic therapy as the deleterious effects are usually limited to the anatomic site of treatment and for a shorter time, whereas systemic therapy often cause functional deficits in several organ systems. the precise information about the quality of life was not available in the records ; hence, it could not be quoted in the study. however, there was qualitative improvement of the patients as mentioned in the data was included in the study. in addition, there was limited use of standard pain assessment scales in the records making the comparisons difficult. shorter courses of hypofractionated prt exemplify common sense end - of - life care and palliation, especially because most patients who are treated for symptom palliation will not survive to face the increased risk of long - term side effects associated with hypofractionated regimens. good clinical judgment and expertise is required to prescribe the correct fractionation schedule for achieving effective symptom palliation with lowest possible cost and inconvenience to the patients and relatives. hypofractionated radiotherapy is a feasible treatment option in patients with advanced incurable disease to achieve effective palliation. | background : palliative radiotherapy (prt) is the eventual requirement in 30 - 50% of all cancer patients. prt is primarily aimed to relieve pain and prevent / treat collapse or fracture in case of bone metastasis, to reduce edema in patients with cranial metastasis, and to control distressing symptoms of rapid primary growth. an audit of prt planned in a busy cancer center can help in the characterization of the requirements of the patients and the formulation of institutional policies.materials and methods : in total, 516 patients who received prt in our regional cancer center from january 2012 to december 2012 and whose complete records were available for analysis were selected for this retrospective study. medical records and radiotherapy files were analyzed to obtain data such as sociodemographic parameters, prescription of prt, and follow up. descriptive statistics were evaluated in terms of frequencies and percentages to allow comparisons.results:of the 516 patients, 73% patients were male ; the median age of the patients receiving prt was 62 years (range 13 - 83 years). about 48% (n = 248) patients received prt at the primary site while rest (52%) were given prt at the metastatic site. the most common indication of prt was pain (56.8% cases), followed by cytostatic prt (19.8%) and raised ict (12.4%). the median dose prescribed was 30 gy (range 8 - 36 gy) delivered in 1 - 12 fractions over the duration of 1 - 18 days. the overall response rate was about 43% at 2 weeks of completion of prt ; the median follow - up of the patients was 154 days (range 9 - 256 days). the long - term symptom relief at median follow up was 8%.conclusions : good clinical judgment and expertise is required in prescribing correct fractionation schedule to achieve effective symptom palliation with lowest possible cost and inconvenience to the patients and relatives. hypofractionated radiotherapy is a feasible treatment option in patients with advanced incurable disease to achieve effective palliation. |
much emphasis has been given to the reduction in coronal caries experience in children. however, the international literature is controversial as to caries rates in adult and elderly populations. some studies demonstrate improved oral health conditions with reduced occurrence of caries and edentulism4,10,15, while others report that geriatric populations have poor oral health status and high caries prevalence, including root caries6,12,18. the occurrence of root caries has been increasingly reported in the last decades, and it has been shown to increase proportionally with age6,12,14,15,17. it should be highlighted that, in some areas, the occurrence of root caries has been considered a public health problem13, and some authors mention root caries as a risk factor for tooth loss in the age group above 60 years5,19. studies have reported the importance of water fluoridation on root caries prevalence8,14,22, yet such studies are scarce in brazil. thus, it is important to investigate the beneficial effect of water fluoridation not only for children, but also for adults and elderly in the brazilian context. therefore, this study investigated the prevalence of root caries in areas with and without water fluoridation at the southeast region of so paulo state, in adult individuals, employees of public and private schools, and elderly population. the research protocol was first reviewed and approved by the institutional review board of so paulo public health school (coep 62/98). this cross - sectional study was conducted at the state of so paulo, southeast region of brazil, in 1998. the study was performed on 29 randomly selected cities, including 16 with fluoridation of public water supply and 13 non - fluoridated cities. these 29 cities corresponded to 4 areas of the regional health directions (dirs) at the regions of campinas (dir xii), piracicaba (dir xv), so joo da boa vista (dir xx) and sorocaba (dir xxiii). random selection of cities according to the presence of water fluoridation was performed according to official records available at the time16. the onset of water fluoridation in the cities ranged from 1972 to 1991 ; most cities initiated water fluoridation in the 1980s. the codes employed for investigation of root caries followed the guidelines of the world health organization22. root caries prevalence was considered as the percentage of people presenting this type of lesion. this condition was also evaluated by mean number of decayed and/or filled roots (df - r), in addition to the mean of each of these components individually, namely mean number of decayed roots (d - r), mean number of filled roots (f - r) and mean number of intact roots despite the presence of gingival recession (s - r). these variables were assessed according to age group (35 - 44 years old and 65 - 74 years old) and gender (male and female). examinations were performed by 33 dental professionals submitted to a 40-hour calibration, including theoretical discussions and practical activities, which simulated the situations experienced during practical work. the consensus technique was adopted, as recommended by the general coordination of the study. inter - examiner agreement for dental caries was evaluated in the southeast region, divided into four areas, revealing the following values : 84%, 96.5%, 96.7% and 99.2%. values above 90% are considered good agreement, thus validating the outcomes ; values of 80% to 89% indicate moderate agreement. the percentage of intra - examiner agreement was calculated during data collection, by reexamination of 10% of the sample. the values obtained were 95.0%, 99.8% and 99.9% ; the results were not obtained for one area. the subjects included elementary school teachers and employees of public and private schools, aged 35 to 44 years old, who were examined at work ; and elderly aged 65 to 74 years old, attending health services or associations, who were examined at these places. individuals were selected by systematic probability sampling (all schools were included in cities with less than 20 schools) and a signed informed consent form was obtained form each participant. elderly were selected among all individuals attending associations or municipal health services from october to december 1998. statistical analysis was performed by mann - whitney test for comparisons among means of df - r and its components (d - r and f - r), s - r and mean number of present teeth according to presence or not of fluoridated water. chi - square test was used to evaluate the mean number of present teeth according to the presence / absence of water fluoridation. table 1 presents the number of dentate individuals examined at the southeast region of so paulo state who were effectively included in the study, according to water fluoridation and gender. variables, such as mean number of present teeth and percentage of missing teeth in the dmft index, were also investigated according to fluoridation of public water supply, as presented in table 2. in the adult group, areas with fluoridated water showed a higher mean number of present teeth and lower mean number of missing teeth. in areas without fluoridated water, the elderly showed higher percentage of missing teeth. regardless of water fluoridation, both adults and elderly presented more missing teeth than women (p<0.05). among the elderly, in areas with fluoridated water, men showed more missing teeth (p<0.001). in the elderly group, only in fluoridated areas, women presented less missing teeth (p<0.001). means followed by different letters in the same line differ in relation to fluoridated water (p < 0.05 ; chi - square test). the prevalence of root caries for the 35 - 44-year - old age group was 17.3% (n=111) and 13.0% (n=53) (p=0.578), respectively, for populations living in cities at the southeast region with or without water fluoridation. for the 65 - 74-year - old age group, the prevalence was 33.8% (n=78) and 29.4% (n=57) (p=0.333), respectively, for fluoridated and non - fluoridated areas. results on the occurrence of root caries relative to water fluoridation and gender are presented in table 3, according to the mean number of decayed and filled roots (df - r), in addition to stratification of df - r components, namely mean number of decayed roots (d - r) and mean number of filled roots (f - r), as well as mean number of exposed yet intact roots (s - r). means followed by different letters in the same line differ in relation to fluoridated water (p < 0.05 ; mann - whitney test).sd= standard deviation. df - r = mean number of decayed and filled roots ; d - r = mean number of decayed roots ; f - r= mean number of filled roots ; s - r= mean number of exposed yet sound roots. in brazil, dental treatment in public health centers prioritizes school age children and the improvement of oral health in this age range, in a general way. on the other hand, adult and elderly patients have a great demand for treatment and often look for tooth extraction, which has a relatively low cost, fast resolution and can be performed in public health centers. this lack of access to preventive care is one of the several factors that can lead to tooth loss in the adult and elderly populations. these two groups can benefit from fluoridated water for control of dental caries, more specifically root caries. however, this benefit may be partial, since many teeth were missing in these individuals. the present cross - sectional study employed secondary data from the oral health epidemiological survey of the state of so paulo 1998, which planned the sample size according to the prevalence of coronal caries ; however, this study investigated root caries. the adult population comprised a specific category of employees and thus the results can not be extrapolated for the southeast region as a whole. since elderly populations usually present relatively low number of remaining teeth, the results of this group should be carefully interpreted because the reduced number of present teeth can underestimate root caries prevalence, considering that more severe cases of root caries might demand tooth extraction. however, in this study, water fluoridation was not a differential factor for edentulism in any age group, unlike the findings of o'mullane,.15 (1996), who reported that both adult and elderly individuals living in fluoridated areas presented lower percentage of edentulism. notwithstanding, the m - t component of the dmft index was higher for individuals living in non - fluoridated areas (table 2), as demonstrated in other studies7,18. this result may imply that, in the southeast region, elderly males (table 3) presented lower root caries prevalence because these individuals had a smaller number of teeth, possibly due to early loss, which may be related to dental caries, especially coronal caries, even though this was not addressed in the present study. it may be assumed that coronal caries led to tooth loss before root caries occurred. in addition to dental caries, tooth loss in these two groups of individuals can be related to several other factors, e.g. periodontal disease, which causes loss of bone support ; dental trauma ; fractures ; and lack of access to oral health services. in this study this point was confirmed by imazato,.9 (2006), who observed that the number of decayed or filled lesions increased as the number of remaining teeth increased. furthermore, the same authors reported that the presence of carious lesions may be related to dry mouth and brushing frequency, as risk factors associated with the presence of root caries. several international studies have reported that the proportion of adults keeping their natural teeth was higher in fluoridated communities compared to non - fluoridated areas3,13,15,18,19, as observed in the present study. marthalerm,.11 (1996) reported a dramatic reduction in the amount of prosthetic services delivered to elderly in fluoridated regions of switzerland, which indirectly represents maintenance of natural teeth by this population. due to the unreliable establishment of the cause of tooth loss in adults, especially elderly, the dmft index is not a safe measurement for caries evaluation as it is for children. consequently, the interpretation of dmft data for adults is difficult, highlighting the need to develop other indices to verify the oral health status in adult patients, especially addressing the root surface. in the present study, the prevalence of root caries was not statistically dependent on the presence of water fluoridation for either of the age groups. conversely, previous studies have reported an inversely proportional relationship between the prevalence of root caries and exposure to fluoridated water1,8,15,19, as also observed for the mean number of filled roots (f - r). additionally, adults living in fluoridated areas had higher mean number of sound roots, i.e. maintenance of intact roots even in the presence of root exposure, which may represent a possible beneficial effect of this method for this age group (table 3). in the same table, among the elderly, it can be observed that the mean of roots caries (d - r) was smaller in areas without fluoridated water, which can be apparently controversial. however, the standard deviation was high for several components of the df - r, and these results can interfere with evaluation of these data. thus, caution is recommended in the analysis of these conditions. when the variables df - r, d - r, f - r and s - r were stratified according to gender, for both adults and elderly, women living in fluoridated areas presented higher mean number of exposed yet intact roots (table 3), which may indirectly suggest a better oral health status in this group. however, a recent study conducted on elderly living in a residential home at turkey20, with mean age of 75 years, did not find difference among df - r and gender. in this study, there was no statistically significant difference between root caries prevalence and gender in any of the age groups. the estimate of root caries prevalence may be impaired by the presence of restorations on this surface, which may have been applied due to caries or not21. however, if only untreated carious lesions are diagnosed, the value of examination is considerably reduced. according to clarkson2 (1995), investigations may underestimate more than two thirds of the prevalence. despite the vulnerability of epidemiological surveys at these age groups, some aspects as the mean number of sound roots and mean number of present teeth were highlighted ; their investigation in studies addressing the tooth root is required. in this study, root caries prevalence was lower in areas with fluoridated water. due to the reduced prevalence of edentulism and increased number of people keeping their natural teeth for a longer period, a future increase in root caries is expected, highlighting the importance of studies related to water fluoridation and its relationship with the oral health of adults and elderly, especially referring to tooth root. | this study aimed to investigate root caries prevalence in areas with and without water fluoridation at the southeast region of so paulo state, in the adult population, employees of public and private schools, and elderly population. epidemiological surveys were conducted according to the world health organization guidelines (1997), including 1,475 dentate individuals aged 35 to 44 years and 65 to 74 years, living in cities representing the southeast of so paulo state, with (n=872) or without (n=603) fluoridated water supply. statistical analysis was performed by mann - whitney and chi - square tests at a significance level of 5%. the prevalence of root caries was 15.6% for the 35 - 44-year - old age group and 31.8% for the 65 - 74-year - old age group. there were no statistically significant differences (p>0.05) in the occurrence of root caries according to water fluoridation, although individuals living at non - fluoridated areas presented higher percentage of missing teeth ; also, there was higher mean number of intact roots at fluoridated areas (p<0.05). most individuals with gingival recession, both adults and elderly, did not have root caries experience. in this study, root caries prevalence was lower in areas with fluoridated water. due to the reduced prevalence of edentulism and increased number of people keeping their natural teeth for a longer period, a future increase in root caries is expected, highlighting the importance of studies related to water fluoridation and its relationship with the oral health of adults and elderly, especially referring to tooth root. |
the general increase in sports - related injuries of the past decades and the high demands for fast and accurate treatment create one of the biggest challenges for the orthopedic surgeon of the 21st century. as such, early osteoarthritis (oa) early oa is considered more difficult to diagnose than oa as signs and symptoms may still be limited, often becoming manifest after higher strains such as sport activities. in contrast, the diagnosis of advanced oa is easily made based on the patient history and physical and x - ray examinations. patients are typically over 50 years old and show symptoms of pain and decreased joint function, which in turn, reduces quality of life. in early oa, the articular cartilage shows fibrillation and vertical fissures that extend into the mid - zone. the articular surface becomes discontinuous and there is progressive increase in subchondral bone plate and subarticular spongiosa. the prevalence of (early) osteoarthritic defects in patients with knee pain (mean age 39 years) has been approximated by widuchowski. other structures such as the menisci and ligaments are frequently affected as well, thus disturbing the joint homeostasis. as early oa generally presents in younger more active patients, traditional approaches for oa such as anti - inflammatory drugs and physical therapy may only provide temporary relief whereas joint replacement limits function and could be prone to future revision surgery. articular cartilage repair may be a promising treatment modality for early oa patients, potentially providing symptom relief and delaying or preventing disease progression. however, there is considerable debate whether cartilage repair is effective in this patient category as the disturbed homeostasis in early oa may create extra difficulty for local cellular regeneration. the aim of this systematic review was to evaluate the existing evidence for treatment of patients with early oa using articular cartilage repair techniques. a systematic review of the literature was conducted on treatment of early oa with articular cartilage repair. the search was performed on november 1, 2012 in the electronic databases of medline, embase, and the cochrane collaboration using the following terms : (osteoarthritis) or (early osteoarthritis) or (moderate osteoarthritis) or (early osteoarthrosis) or (osteoarthritic) or (early osteoarthritic changes) or (early arthritis) or (degenerative) or (salvage) and ((cartilage repair) or (tissue engineering) or (cartilage restoration) or (autologous chondrocyte implantation) or (autologous chondrocyte transplantation) or (matrix - assisted autologous chondrocyte transplantation) or (matrix - induced autologous chondrocyte implantation) or (mact) or (maci) or (characterized chondrocyte implantation) or (autologous osteochondral transplantation) or (osteochondral autologous transplantation) or (oats) or (osteochondral autograft transplantation) or (oct) or (mosaicplasty) or (allograft osteochondral transplantation) or (scaffolds) or (microfracture) or (microfracturing). inclusion criteria for relevant articles that were used during screening of titles and abstracts included : therapeutic studies of articular cartilage repair in patients with early osteoarthritis and/or degenerative changes with a minimal follow - up of 12 months. etiologic studies, reviews, case reports, animal and cadaver studies were excluded and also articles of which the full texts were not retrievable. the cochrane handbook for systematic reviews and the guidelines for transparent reporting of systematic reviews and meta - analysis (prisma) were used. to assess the methodological quality, 2 independent observers (tdw and lv) appraised each study using the modified coleman methodology score. a score of 100 represents a study with a solid design and no biases or confounding factors. if there was a difference in scores, studies were discussed and a consensus was reached. extracted data from the selected studies included patient demographics, grade of oa, surgical procedure(s), defect sizes, and clinical outcome scores. a systematic review of the literature was conducted on treatment of early oa with articular cartilage repair. the search was performed on november 1, 2012 in the electronic databases of medline, embase, and the cochrane collaboration using the following terms : (osteoarthritis) or (early osteoarthritis) or (moderate osteoarthritis) or (early osteoarthrosis) or (osteoarthritic) or (early osteoarthritic changes) or (early arthritis) or (degenerative) or (salvage) and ((cartilage repair) or (tissue engineering) or (cartilage restoration) or (autologous chondrocyte implantation) or (autologous chondrocyte transplantation) or (matrix - assisted autologous chondrocyte transplantation) or (matrix - induced autologous chondrocyte implantation) or (mact) or (maci) or (characterized chondrocyte implantation) or (autologous osteochondral transplantation) or (osteochondral autologous transplantation) or (oats) or (osteochondral autograft transplantation) or (oct) or (mosaicplasty) or (allograft osteochondral transplantation) or (scaffolds) or (microfracture) or (microfracturing). inclusion criteria for relevant articles that were used during screening of titles and abstracts included : therapeutic studies of articular cartilage repair in patients with early osteoarthritis and/or degenerative changes with a minimal follow - up of 12 months. etiologic studies, reviews, case reports, animal and cadaver studies were excluded and also articles of which the full texts were not retrievable. the cochrane handbook for systematic reviews and the guidelines for transparent reporting of systematic reviews and meta - analysis (prisma) were used. to assess the methodological quality, 2 independent observers (tdw and lv) appraised each study using the modified coleman methodology score. a score of 100 represents a study with a solid design and no biases or confounding factors. if there was a difference in scores, studies were discussed and a consensus was reached. extracted data from the selected studies included patient demographics, grade of oa, surgical procedure(s), defect sizes, and clinical outcome scores. cochrane resulted in 226 and 26 articles, respectively. after excluding duplicates, 1,212 articles remained for screening title and abstract. one full - text article could not be retraced and was therefore excluded (fig. all included articles were case series reporting on a total of 502 patients (mean age range 36 - 57 years). one report presented the 4-year follow - up of a subset of patients from one of the included studies. all studies were of level 4 evidence according to the oxford center for evidence - based medicine and had a mean modified coleman score of 58 (range = 36 - 73 ; table 1). study design and quality assessment. note : cs = case series ; coleman = modified coleman score. minas. performed a prospective study including 153 patients with early oa who were treated with first - generation aci and followed for a mean of 64 months (range = 24 - 132 months). in this study, radiographic criteria for early oa were peripheral intra - articular osteophyte formation and/or 0% to 50% joint space narrowing as defined by ahlbck stage 0-i whereas the clinical criteria included evidence of bipolar (kissing) lesions or generalized chondromalacia. concomitant procedures were performed in 103 (66.5%) patients (table 2). at 5 years, 92% of patients experienced improvement in the womac (western ontario and mcmaster universities osteoarthritis index) score (p < 0.001) and were able to delay the need for joint replacement. of the patients, 8% were considered treatment failures and were revised to partial (n = 2) or total (n = 10) joint arthroplasty (table 3). study and patient characteristics. note : aci = autologous chondrocyte implantation ; n / a = not applicable ; mf = microfracture ; cf = carbon fibers. previous procedures : debridement, chondroplasty, marrow stimulation, ligament reconstruction, and menisectomy. note : tka = total knee arthroplasty ; aci = autologous chondrocyte implantation ; womac = western ontario and mcmaster universities osteoarthritis index ; ikdc = international knee documentation committee knee examination form ; koos = knee injury and osteoarthritis outcome score ; cincinnati = cincinnati knee rating system ; mf = microfracture ; cf = carbon fibers ; vas = visual analogue scale ; tegner = tegner activity scale ; n / a = not applicable. in 2 similar case series, filardo. evaluated 57 patients with degenerative grade iii - iv icrs (international cartilage repair society) lesions (at least 6 months of symptoms not responding to conservative treatment) and 44 patients with cartilage lesions in oa knees (kellgren lawrence grade 2 - 3) who had refused total knee arthroplasty (tka). in the first study, patients were treated with second - generation aci and followed for a mean of 74 months. after 6 years, 85% of patients showed a significant improvement in the international knee documentation committee (ikdc) score (p < 0.005). ten patients (18.5%) required reintervention because of symptoms related to the primary defect (table 3). in the second study, 44 patients received the same treatment (hyalograft c) and were followed for up to 9 years. although clinical improvement was shown (ikdc p < 0.0005), the failure rate was higher than the previous study (27.3% vs. 18.5% ; table 3). ossendorf. used second - generation aci to treat 40 patients suffering from chronic posttraumatic and/or degenerative cartilage lesions. the mean defect size was 5.0 cm and the follow - up was 24 months (table 2). comparable clinical improvement (koos / ikdc p < 0.05) in both the osteoarthritic and posttraumatic / mild degenerative groups was seen. second - look biopsies of 4 patients showed hyaline - like and mixed repair tissues. at final follow - up, 22 patients had radiological oa (kellgren lawrence score 2) while still showing significant improvement (p < 0.0001) in the lysholm and cincinnati knee rating systems. later presented the 4-year clinical improvement (lysholm and koos p < 0.05) of 19 patients of the cohort of ossendorf. who preoperatively had confirmed oa with a kellgren mri analysis showed defect filling in 16 out of these 19 patients. in a case series of 56 patients aged 45 years, rosenberger. used first - generation aci to treat 32 patients with large early osteoarthritic lesions (mean size 11.7 cm). the same criteria for early oa as described by minas. were used. overall, patients rated their outcomes as excellent (72%) and comparable clinical improvement was seen for all defect categories, that is, simple, complex, and salvage. both failure of treatment and conversion to tka occurred in 3 patients (9% ; table 3). hollander. compared second - look biopsies from 14 aci patients with no x - ray signs of oa (ahlbck score 0) and 9 patients with x - ray signs of oa (ahlbck score iii - iv). after 1 year, the repair tissue showed hyaline cartilage in 67% of oa joints compared with 36% of non - oa joints. two out of 3 biopsies from patients with advanced oa (ahlbck iv) showed hyaline cartilage. evaluated 44 patients with an average lesion size of 3.9 cm (range = 1 - 6 cm, outerbridge grade iv) with moderate osteoarthritic changes who underwent microfracture (mf ; table 2). after a mean of 2.3 years, significant improvement (p < 0.05) in pain and daily living was seen and 95% of patients were rated good to excellent (table 3). in addition, using second - look arthroscopy, defect filling was determined which was confirmed with histologic evaluation and type ii collagen staining. used drilling and subsequent carbon fiber scaffold implantation for treatment of early osteoarthritic defects in 2 separate cohorts with a short - term success rate of more than 80% in terms of pain and clinical outcome (tables 2 and 3). all patients had icrs grade iii - iv lesions and fulfilled the criteria for symptomatic oa by the subcommittee of the american college of rheumatology. cochrane resulted in 226 and 26 articles, respectively. after excluding duplicates, 1,212 articles remained for screening title and abstract. one full - text article could not be retraced and was therefore excluded (fig. all included articles were case series reporting on a total of 502 patients (mean age range 36 - 57 years). one report presented the 4-year follow - up of a subset of patients from one of the included studies. all studies were of level 4 evidence according to the oxford center for evidence - based medicine and had a mean modified coleman score of 58 (range = 36 - 73 ; table 1). study design and quality assessment. note : cs = case series ; coleman = modified coleman score. minas. performed a prospective study including 153 patients with early oa who were treated with first - generation aci and followed for a mean of 64 months (range = 24 - 132 months). in this study, radiographic criteria for early oa were peripheral intra - articular osteophyte formation and/or 0% to 50% joint space narrowing as defined by ahlbck stage 0-i whereas the clinical criteria included evidence of bipolar (kissing) lesions or generalized chondromalacia. concomitant procedures were performed in 103 (66.5%) patients (table 2). at 5 years, 92% of patients experienced improvement in the womac (western ontario and mcmaster universities osteoarthritis index) score (p < 0.001) and were able to delay the need for joint replacement. of the patients, 8% were considered treatment failures and were revised to partial (n = 2) or total (n = 10) joint arthroplasty (table 3). study and patient characteristics. note : aci = autologous chondrocyte implantation ; n / a = not applicable ; mf = microfracture ; cf = carbon fibers. previous procedures : debridement, chondroplasty, marrow stimulation, ligament reconstruction, and menisectomy. note : tka = total knee arthroplasty ; aci = autologous chondrocyte implantation ; womac = western ontario and mcmaster universities osteoarthritis index ; ikdc = international knee documentation committee knee examination form ; koos = knee injury and osteoarthritis outcome score ; cincinnati = cincinnati knee rating system ; mf = microfracture ; cf = carbon fibers ; vas = visual analogue scale ; tegner = tegner activity scale ; n / a = not applicable. in 2 similar case series, filardo. evaluated 57 patients with degenerative grade iii - iv icrs (international cartilage repair society) lesions (at least 6 months of symptoms not responding to conservative treatment) and 44 patients with cartilage lesions in oa knees (kellgren lawrence grade 2 - 3) who had refused total knee arthroplasty (tka). in the first study, patients were treated with second - generation aci and followed for a mean of 74 months. after 6 years, 85% of patients showed a significant improvement in the international knee documentation committee (ikdc) score (p < 0.005). ten patients (18.5%) required reintervention because of symptoms related to the primary defect (table 3). in the second study, 44 patients received the same treatment (hyalograft c) and were followed for up to 9 years. of the patients, although clinical improvement was shown (ikdc p < 0.0005), the failure rate was higher than the previous study (27.3% vs. 18.5% ; table 3). used second - generation aci to treat 40 patients suffering from chronic posttraumatic and/or degenerative cartilage lesions. thirteen patients had early oa with a jger wirth score of 3 whereas 27 patients had a jger the mean defect size was 5.0 cm and the follow - up was 24 months (table 2). comparable clinical improvement (koos / ikdc p < 0.05) in both the osteoarthritic and posttraumatic / mild degenerative groups was seen. second - look biopsies of 4 patients showed hyaline - like and mixed repair tissues. at final follow - up, 22 patients had radiological oa (kellgren lawrence score 2) while still showing significant improvement (p < 0.0001) in the lysholm and cincinnati knee rating systems. later presented the 4-year clinical improvement (lysholm and koos p < 0.05) of 19 patients of the cohort of ossendorf. who preoperatively had confirmed oa with a kellgren mri analysis showed defect filling in 16 out of these 19 patients. in a case series of 56 patients aged 45 years, rosenberger. used first - generation aci to treat 32 patients with large early osteoarthritic lesions (mean size 11.7 cm). the same criteria for early oa as described by minas. were used. overall, patients rated their outcomes as excellent (72%) and comparable clinical improvement was seen for all defect categories, that is, simple, complex, and salvage. both failure of treatment and conversion to tka occurred in 3 patients (9% ; table 3). hollander. compared second - look biopsies from 14 aci patients with no x - ray signs of oa (ahlbck score 0) and 9 patients with x - ray signs of oa (ahlbck score iii - iv). after 1 year, the repair tissue showed hyaline cartilage in 67% of oa joints compared with 36% of non - oa joints. two out of 3 biopsies from patients with advanced oa (ahlbck iv) showed hyaline cartilage. bae. evaluated 44 patients with an average lesion size of 3.9 cm (range = 1 - 6 cm, outerbridge grade iv) with moderate osteoarthritic changes who underwent microfracture (mf ; table 2). after a mean of 2.3 years, significant improvement (p < 0.05) in pain and daily living was seen and 95% of patients were rated good to excellent (table 3). in addition, using second - look arthroscopy, defect filling was determined which was confirmed with histologic evaluation and type ii collagen staining. used drilling and subsequent carbon fiber scaffold implantation for treatment of early osteoarthritic defects in 2 separate cohorts with a short - term success rate of more than 80% in terms of pain and clinical outcome (tables 2 and 3). all patients had icrs grade iii - iv lesions and fulfilled the criteria for symptomatic oa by the subcommittee of the american college of rheumatology. early oa is increasingly being recognized in patients who wish to remain active while not accepting the limitations of conservative treatment or joint replacement. as such, the research field in articular cartilage repair is extending its grounds, aiming at early treatment and prevention of disease progression. this review evaluated the literature that describes the use of cartilage repair procedures in patients with early oa. after application of inclusion and exclusion criteria, 9 articles of generally low methodological quality (mean coleman score 58) could be included. the low methodological quality and heterogeneity in patient populations is a known limitation of clinical research in cartilage repair. this heterogeneity may even be more profound in early oa, as these patients frequently have been subjected to multiple previous and/or concomitant procedures. in fact, in the included studies, 46% to 100% of patients received a previous treatment for to the index knee and up to 67% of patients underwent concomitant procedures (table 2). the use of concomitant treatments in this patient category does not seem to have a negative effect on clinical outcome as no substantial differences in outcome were reported. this is surprising, as it is thought that concurrent injuries can disturb joint homeostasis. for example, anterior cruciate ligament and meniscal tears are known to be a risk factor for oa. indeed, in one of the included studies by filardo. moreover, numerous studies have found correlation between time since symptom onset and clinical outcome, suggesting a disturbance in joint homeostasis could affect cartilage regeneration. the combined treatment of articular cartilage defects and malalignment is increasingly being applied and shows promising results. bauer. demonstrated significant clinical improvement in 18 patients (mean age = 47 years) with medial knee oa receiving matrix - induced aci and a high tibial osteotomy. found a 91% survivorship 7 years after combined high tibial osteotomy and mf in 106 patients. twelve patients (11%) required tka after a mean of 81 months. in our review, we found a conversion to tka rate of 2.5% to 6.5% for aci (table 3). this is encouraging, as the conversion rates for isolated osteotomy has been found to be higher (20% to 50%). it should be acknowledged, however, that only a randomized controlled trial would be able to distinguish between the effect of cartilage repair and realignment osteotomy. of all patients included in this review, this is not surprising, as in general, aci is preferred in larger and complex lesions and clinical outcome after mf may have the tendency to deteriorate over time. indeed several in vitro studies have shown good proliferation of oa chondrocytes and recently, aci showed superior macroscopic and histological results compared to cell - free approaches in an early oa model in vivo. while bae. used mf to treat cartilage defects in early oa, no patients underwent previous procedures and the mean defect size was smaller (3.9 cm) than in most studies (table 2).. recently showed that tissue removed prior to tka in failed mf patients with early oa consisted of fibrocartilaginous tissue. this could imply that the quality of the regenerative tissue is an important prognostic factor. however, as brittberg. and de windt. also demonstrated clinical improvement using marrow stimulation, long - term (comparative) research is needed to determine if regenerative tissue quality affects clinical outcome in early oa. although a study aiming specifically at osteochondral autograft or allograft transfer for early oa could not be included, these procedures may well be an option for future clinical trials in this patient category. performed osteochondral autograft transfer in 82 professional athletes including those with early signs of oa and found similar success rates compared with that of less athletic patients, although high motivation resulted in better subjective evaluation. osteochondral allograft transfer has successfully been applied in young patients with steroid - induced osteonecrosis showing 90% graft survival at 6 years. at their last follow - up in contrast, beaver. found a higher failure rate for posttraumatic osteoarticular bipolar lesions treated with fresh allografts.. limitations of this review are the low methodological quality, small sample sizes, and heterogeneity of patients. although the inclusion criteria were strictly aimed at early oa, the variation in the definition of early oa may also limit the findings of this review. primarily used the radiographic ahlbck score whereas ossendorf. and kreuz. all others applied clinical and macroscopic grading only. in recognizing and defining early oa as a disease entity, the osteoarthritis research society international histologic histochemical grading system was recently adapted aiming specifically at early oa the development and use of such a standardized radiological and clinical grading system could especially be important for the comparability between (future) clinical trials in early oa. the excellent clinical outcome (up to 4 years) in a small subset of patients with advanced oa as shown by ossendorf. and are promising findings, which could imply that cartilage repair can be an alternative for active patients with advanced oa. furthermore, the comparable failure rates in early oa compared with the general population, that is, 8% to 27.3% versus 1.5% to 33%, respectively, underline the potential of cartilage repair as a salvage procedure. however, it is important to note that filardo. found a relatively high failure rate, which increased for the longer follow - up. although they still consider aci as an option to delay joint replacement, these findings stress the importance of careful treatment selection and patient counseling. in considering tka as an alternative for younger patients with oa, keeney. performed a systematic review and found surgeon - measured clinical improvement in patients younger than 55 years who had received tka. in this study, a moderate increase in second decade implant failures (from 91% to 99% implant survival to 85% to 97%) was found. unfortunately, a lack of patient - based and functional outcome measures as well as the difference in patient selection makes these results difficult to compare with the results of this review. thus, a (randomized) comparison between tka, cartilage repair, and/or conservative measures for early oa could be of great value. nevertheless, revisions and the known limitations in activity following tka emphasize the need for different treatment options in active patients with (early) oa. in conclusion, although not yet a fact, there is low - level evidence that suggests cartilage repair is a feasible treatment for (early) oa, which demonstrates preliminary effectiveness of up to 9 years. although a (randomized controlled) trial in this patient category with long - term follow - up is needed, the literature suggests aci could provide short- to mid - term clinical outcome and delay the need for tka. the use of standardized criteria for early oa and implementation of (randomized) trials with long - term follow - up may allow for further expansion of the research field in articular cartilage repair to the challenging population with (early) oa. | early osteoarthritis (oa) is increasingly being recognized in patients who wish to remain active while not accepting the limitations of conservative treatment or joint replacement. the aim of this systematic review was to evaluate the existing evidence for treatment of patients with early oa using articular cartilage repair techniques. a systematic search was performed in embase, medline, and the cochrane collaboration. articles were screened for relevance and appraised for quality. nine articles of generally low methodological quality (mean coleman score 58) including a total of 502 patients (mean age range = 36 - 57 years) could be included. in the reports, both radiological and clinical criteria for early oa were applied. of all patients included in this review, 75% were treated with autologous chondrocyte implantation. good short - term clinical outcome up to 9 years was shown. failure rates varied from 8% to 27.3%. the conversion to total knee arthroplasty rate was 2.5% to 6.5%. although a (randomized controlled) trial in this patient category with long - term follow - up is needed, the literature suggests autologous chondrocyte implantation could provide good short- to mid - term clinical outcome and delay the need for total knee arthroplasty. the use of standardized criteria for early oa and implementation of (randomized) trials with long - term follow - up may allow for further expansion of the research field in articular cartilage repair to the challenging population with (early) oa. |
systemic lupus erythematosus (sle) is a common autoimmune disease characterized by a dysregulation of the immune system that leads to chronic systemic inflammation [1, 2 ]. as previous reported, sle is associated with accelerated atherosclerosis and increased risk of cardiovascular events driving high morbidity and mortality rates [3, 4 ]. however, the endothelial damage, accelerated atherosclerosis, and increased cardiovascular disease observed in sle patients can not be fully explained by traditional cardiovascular risk factors or the use of corticosteroids. it has been proposed that systemic inflammation, dysregulated cytokine profile, and altered t cell subsets play important roles in endothelial dysfunction and increased cardiovascular risk presenting in sle patients. therefore, identification of cardiovascular damage and repair biomarkers related to sle may reveal insights into pathogenesis and may be used to monitor the cardiovascular risk and improve the cardiovascular health. recently, a specific t cell population, termed angiogenic t cells (tang), has been described that promotes the formation of new blood vessels and repair of damaged endothelium by cooperating with endothelial progenitor cells (epcs). furthermore, hur and colleagues showed that tang cells are characterized by the expression of cd3, the platelet endothelial cell adhesion molecule (cd31), and the receptor for stromal - derived factor 1 (cxcr4). as a subset of cd3 + t cells, tang cells also express cd4 or cd8. it has been reported that decreased tang cell frequencies are associated with vascular disease, and circulating tang cells are decreased in patients with ra, an autoimmune disease that has increased prevalence of cardiovascular events. however, little was known about the percentages of tang cell subsets (including cd3+cd31+cxcr4 +, cd4+cd31+cxcr4 +, and cd8+cd31+cxcr4 + cells) in circulation of sle patients. therefore, we explore whether the percentages of tang cell subsets measured by flow cytometry are different in patients with sle comparing with healthy controls (hc). samples of peripheral blood (pb) were obtained from 41 sle patients and 22 hc. disease activity was assessed by systemic lupus erythematosus disease activity index (sledai) score at the time of recruitment. none of the patients had a history of coronary artery disease, diabetes, or cardiac insufficiency. other information of patients ' demographic, clinical, and immunological manifestations and current medications is described in table 1. this study was approved by the ethics committee of xijing hospital and conforms to the recommendations of the declaration of helsinki. pb mononuclear cells (pbmcs) were isolated from sodium heparinized whole blood by ficoll - paque density gradient centrifugation (ge healthcare, pittsburgh, pa, usa). briefly, pbmcs were stained with the following fluorochrome conjugated monoclonal antibodies : anti - cd3-peridin chlorophyll protein (percp), anti - cd4-percp, anti - cd8-percp, anti - cd31-phycoerythrin (pe), anti - cxcr4-allophycocyanin (apc), and isotype - matched control igg antibodies (all from bd biosciences, san diego, ca, usa) for 30 min at room temperature, according to the manufacturer 's instructions. after being washed with pbs, a minimum of 20,000 events per tube was acquired using a facscalibur flow cytometer (bd biosciences) and analyzed using cellquest software (bd biosciences) and flowjo 7.6.1 software (tree star). for all tests, a two - sided p value less than 0.05 was considered significant. data analyses were performed using spss 17.0 software (spss, chicago, il, usa). a total of 42 patients with sle and 22 hc were recruited into the study. the traditional cardiovascular risk factors have been reported to have an inverse relationship with the circulating tang cells. in this study, the group of sle patients and hc did not differ significantly in cardiovascular risk factors, including smoking, diabetes, hyperlipidemia, and hypertension (all p > 0.05). as shown in figure 1(a), representative examples of cd31 and cxcr4 expression in cd3 +, cd4 +, and cd8 + t cells were analyzed by flow cytometry. the percentages of circulating cd31+cxcr4 + cells in total cd3 + t cells (tang) and cd31+cxcr4 + cells in cd4 + t cells (cd4 + tang) tended to be slightly higher in sle patients when compared to hc ; however, the differences did not reach statistical significance (figure 1(b)). what is more, the percentages of cd31+cxcr4 + cells in cd8 + t cells (cd8 + tang) were significantly increased in patients with sle as compared to hc (figure 1(b)). then, we wanted to determine whether alterations in the percentages of circulating tang cell subsets were associated with autoantibody status in sle patients. the most striking observation came after subdividing sle patients into two groups based on the anti - dsdna antibody status. the percentage of tang, cd4 + tang, and cd8 + tang in anti - dsdna - positive sle patients was significantly increased when compared with their negative counterparts and hc (figure 2(a)). and the percentage of tang and cd8 + tang in anti - dsdna - positive, anti - ssa / ssb - positive, or anti - sm - positive sle patients was significantly increased as compared to hc. however, no differences in circulating tang cell subsets were observed between anti - ssa / ssb - positive or anti - sm - positive sle patients and their negative counterparts (figures 2(b) and 2(c)). the correlation of circulating tang cell subsets with clinical features in sle patients was shown in table 2. in order to assess the relationship between tang cell subsets and disease activity in sle, we performed a correlation between the percentages of tang, cd4 + tang, cd8 + tang, and sledai scores. no significant correlation was found between tang cell subsets and sledai scores (table 2). additionally, the percentages of circulating tang, cd4 + tang, and cd8 + tang were negatively correlated with age at sampling and age at diagnosis, but not disease duration (table 2). growing evidences indicate that angiogenesis and angiogenic factors critically contribute to the pathogenesis of sle [1214 ]. indeed, in vitro and in vivo experiments showed that tang cells were required for colony formation and differentiation of early epcs and were supposed to stimulate the endothelial cells by secreting angiogenic cytokines. furthermore, tang cells enhanced endothelial cell proliferation and function, suggesting that tang cells may be viewed as a biomarker for cardiovascular risk. therefore, in this study, the percentages of circulating tang cell subsets, including tang, cd4 + tang, and cd8 + tang, were comprehensively assessed by flow cytometry in sle patients. similar to a recent study, our data showed that the percentage of circulating tang cells in sle patients was not different from hc. lopez. [10, 15 ] showed that the percentage of tang cells is only about 8% in hc, ra, and sle patient. in agreement with the data of a recent study, we showed that the circulating tang level was about 3040% in hc and sle patients. in fact, the population studied in our and kim. work comprised asians, whereas the studies of lopez. we believe that further investigations with a larger number of patients and with follow - up observation are needed to confirm and extend the current results. meanwhile, we found that not only cd3 + cells, but also cd4 + and cd8 + t cells express cd31 and cxcr4 on their surface. thus, the levels of circulating cd4 + tang and cd8 + tang cells were also examined in this study. there were no significant differences in the percentage of cd4 + tang cells between sle patients and hc. however, the percentage of circulating cd8 + tang cells was significantly increased in sle patients when compared to hc, suggesting enhanced cd8 + tang cells may be a repair response to the endothelial damage present in sle patients. in addition, a defective functionality has been reported about tang cells from sle patients, including cytotoxic phenotype and accelerated senescence. as previously reported, several autoantibodies, including anti - dsdna, anti - ssa / ssb, and anti - sm antibodies, play important roles in the diagnosis, disease activity evaluation, organ involvement, and prognosis evaluation of sle. moreover, it has been reported that autoantibody status is associated with vasculopathy and cardiovascular risk in sle and other diseases [1820 ]. in the present work, among sle patients with positive antibodies, both the tang and cd8 + tang cell percentages were significantly increased as compared to hc. interestingly, after dividing these patients according to the presence or absence of antibodies, anti - dsdna - positive sle patients, but not anti - ssa / ssb - positive or anti - sm - positive patients, demonstrated increased levels of tang, cd4 + tang, and cd8 + tang cells as compared to their negative counterparts and hc. therefore, these data indicate that anti - dsdna - positivity may isolate a subset of sle patients associated with endothelial damage and at higher risk of vasculopathy. we did not find any statistically significant correlations between tang cell subsets and disease duration or sle activity according to sledai. as reported in the literatures [7, 10, 15 ], tang cell levels were correlated inversely with age in hc, ra, and sle patients. in line with these results, we showed that tang and their subpopulations cd4 + tang and cd8 + tang cell levels were negatively correlated with age at sampling and age at diagnosis. these findings supported that late age is associated with defective endothelial repair and cardiovascular risk, and tang cell subsets may be used as a biological marker for cardiovascular disease. our results demonstrated that the level of cd8 + tang is elevated in sle patients comparing with hc. and the presence of autoantibody, especially for anti - dsdna antibodies, may identify a group of sle patients with increased tang cell subsets and higher risk of vasculopathy. these findings suggest that, for the first time, circulating cd8 + tang cells may be viewed as a potentially useful biomarker of endothelial damage in sle. further studies are required to clarify the function of cd8 + tang cells and investigate the mechanism for their change in sle. | objective. systemic lupus erythematosus (sle) is associated with accelerated atherosclerosis and increased cardiovascular risk. angiogenic t cells (tang), a specific t cell subset, have been identified and involved in the repair of damaged endothelium. this study aimed to analyze the tang cell subsets in relation to disease specific features from sle patients. methods. tang cell subsets were assessed in peripheral blood samples from 41 sle patients and 22 healthy controls (hc) by flow cytometry on the basis of cd31 and cxcr4 expression on cd3 +, cd4 +, and cd8 + t cells. results. the percentage of circulating cd8+cd31+cxcr4 + t cells (cd8 + tang), but not cd3+cd31+cxcr4 + t cells (tang) and cd4+cd31+cxcr4 + t cells (cd4 + tang), in sle was higher than hc. the percentages of tang cell subsets in anti - dsdna - positive sle patients were significantly increased as compared to their negative counterparts and hc. additionally, the levels of circulating tang cell subsets were negatively correlated with age at sampling and at diagnosis, but not disease duration or disease activity. conclusion. anti - dsdna - positivity may identify a group of sle patients with increased tang cell subsets and circulating cd8 + tang cells may be viewed as a potentially useful biomarker of endothelial damage and cardiovascular risk in sle. |
the recent widespread use of contrast - enhanced computed tomography (ct) or magnetic resonance imaging has contributed to increasing the detection of small renal masses (srms) [1 - 3 ]. incidentally discovered srms are typically low - stage, slow - growing masses with low malignancy potential. thus, the standard treatment for a srm has shifted from radical nephrectomy to partial nephrectomy (pn), which has been shown to confer equivalent oncologic and functional outcomes to those of radical nephrectomy for patients with renal tumors smaller than 4 cm. lucas. reported that radical nephrectomy carries seven times the risk of developing stage 3 chronic kidney disease as that in similar patients undergoing pn or radiofrequency ablation (rfa). along with the developments of minimally invasive approaches, laparoscopic pn has been reported to have oncological efficacy comparable to that of open pn (opn). laparoscopic pn requires high laparoscopic dexterity, however, and even for those with experience requires a longer ischemic time and is associated with more complications than opn. thus, current treatment guidelines recommend the use of thermal ablative therapies for the primary treatment of srms for older patients, those with significant medical comorbidities who are poor surgical candidates, those with genetic predispositions to recurrent tumor formation, and those with imperative indications for pn procedures. the basis for these recommendations is the lack of long - term oncologic efficacy data, the unreliability of measures of treatment efficacy, and the higher rates of local recurrence compared with surgery in the setting of recurrence. the potential benefits of ablative techniques are reduced perioperative morbidity, shorter hospital stay, faster recovery, and preservation of renal function. surgical margins are not considered as a treatment endpoint, which further underscores how the principles defining successful ablation differ from those for surgical extirpation. since then, many reports have been published on rfa for srms, which have shown favorable outcomes in terms of local tumor control [14 - 17 ]. in laparoscopic rfa, the kidney surrounding the tumor is exposed and the perirenal fat covering the tumor is removed and sent for pathology. a steerable laparoscopic ultrasound probe is introduced to visualize the tumor size and location. the electrode probe is placed in the deepest part of the renal tumor under real - time laparoscopic ultrasound guidance. we have been performing both opn and laparoscopic rfa on selected patients since january 2007 and have been researching these patients with serial laboratory assessments and imaging tools such as ct. this study was performed with long - term oncologic data for the purpose of evaluating oncologic outcomes and renal function status at a minimum follow - up of 3 years. since january 2007, 55 patients with exophytic solitary srms were treated with either opn or laparoscopic rfa by a single surgeon. laparoscopic rfa was performed in a transperitoneal or retroperitoneal approach according to tumor location, whereas pn was performed in a retroperitoneal approach. all cases of laparoscopic rfa had undergone preablative biopsy. however, we did not perform frozen biopsy. the choice of approach was based on tumor location, clinical judgment, or patient preference ; rfa was primarily indicated as a treatment for an exophytic renal mass of less than 4 cm in the greatest dimension. the use of rfa was primarily considered in contraindicated patients who had an endophytic tumor, because injury to the collecting system could result. ureteral protection should be considered when laparoscopic rfa is to be performed in cases of an endophytic tumor near the pelvis and ureter. thus, the indications for operative laparoscopic rfa were as follows : 1) cases in which the greatest dimension of the renal mass was <3 cm, 2) cases in which the collecting system and renal calyx were free from the tumor margins by 1 cm, and 3) cases in which the great vessels were free from the tumor margins by 1 cm. rfa was performed with a 200 w generator (radionics, burlington, ma, usa) and a single (with one 3.0-cm tip) internally cooled electrode (radionics, burlington, ma, usa) with an impedance - controlled pulsed current. the ablation time was a maximum of 12 min for one cycle, and the ablation cycle was repeated if the target temperature achieved was suboptimal. on the basis of the size and location of the tumor, overlapping ablations were performed in some patients by repositioning the electrode to completely ablate the entire tumor. the follow - up for each patient included chest radiography, laboratory tests, and ct. for evaluation of therapeutic efficacy, the absence of enhancement inside the tumor was taken to indicate technical success. a follow - up ct was conducted at intervals of 1, 3, and 6 months and then every 6 months over the years. one - month follow - up ct was performed to determine whether there was any remnant or residual enhancement of the ablated lesion. the technical success rate was defined as complete ablation of the tumor following the initial procedure or additional sessions with a 1-month follow - up. recurrence was defined as growth of the tumor or any new enhancing portions at 3 months after confirmed nonenhancement of the initial rfa lesion. mann - whitney u test, pearson 's chi - square test, and repeated - measures analysis of variance between the two groups were used for analysis of the characteristics of each group. a p - value statistical analyses were performed by using ibm spss ver. 18.0 (ibm co., armonk, ny, usa). the patients ' mean age, the mean follow - up time, body mass index, baseline serum creatinine, and baseline serum hemoglobin did not differ significantly between the two groups. the maximum tumor diameter (2.31.27 cm) in the laparoscopic rfa group was similar to that in the opn group (2.40.79 cm, p=0.07). the estimated blood loss (ebl) in the laparoscopic rfa group (40.520 ml) was significantly lower than in the opn group (64.845 ml, p=0.03). the operation time (103.2728.36 minutes) in the laparoscopic rfa group was significantly shorter than in the opn group (148.6440.86 minutes, p=0.04).the mean hospital stay (8.333.23 days) in the laparoscopic rfa group was significantly shorter than in the opn group (12.283.29 days, p=0.00). pathologic results with renal cell carcinoma were slightly lower in the laparoscopic rfa group (31/41, 75.61%) than in the opn group (12/14, 85.71% ; p=0.42). no recurrence or metastasis was seen in either group. during the mean follow - up period of 50 months, radiologic evidence of incomplete ablation one month after the initial laparoscopic rfa, an 86-year - old patient had an enhanced remaining tumor by ct. the patient had no findings of recurrence on radiological follow - up after 12 months. the case was in the laparoscopic rfa group (1/41, 2.44%). in march 2007, after 1 month, the patient had an upper ureteral stricture. at the 3-month follow - up ct, the patient had renal shrinkage. no patients in either group had minor complications such as transfusion, atelectasis, or wound infection. the postoperative serum creatinine (0.880.29) in the laparoscopic rfa group was similar to that in the opn group (0.890.32, p=0.87). concerning the effect of both groups on preserving renal function, creatinine clearance according to cockcroft and gault equation levels in the laparoscopic rfa group was not significantly different from that in the opn group (p=0.31), as shown in table 3. one of the major reasons for this guideline recommendation by the american urological association and the european association of urology is the lack of long - term oncologic efficacy data for rfa compared with opn. rfa was initially introduced to treat selected patients who had high surgical or anesthetic risk with a solitary tumor or multifocal renal tumors. various reports on local tumor control have so far been promising [14 - 17 ]. the outcomes of rfa are affected by the following factors : tumor size and location, tissue impedance, ablation time, amount of energy delivered, and surface area of the electrodes. less than 3 cm after the initial rfa but reported a noticeably smaller response rate with tumors greater than 5 cm in size. also, lucas. reported that rfa is superior to pn in terms of preserving renal function in patients with srms. the criteria of therapeutic response were based on the report by goldberg.. complete response is defined as the absence of any enhancement within the tumor as observed in the preoperative contrast - enhanced ct image. a benign periablation enhancement, which can measure up to 12 mm, typically suggests a transient benign physiologic response to a thermal injury and may persist for up to 3 months after the ablation. on the other hand, the local recurrence rate varies from 0% to 11.1% in cases in which technical success is achieved during the initial rfa. rfa is known to cause coagulation necrosis within the tumor by the following mechanism. when electrical current from the uninsulated rf electrode is delivered to the tumor, ionic agitation occurs in the tissue, resulting in heat energy. it is reported that the ablative effect on a centrally located tumor is lower because of the heat sink effect of central blood vessels near the renal hilum, in which regional vascular flow reduces the extent of the thermally induced coagulation. by contrast, the ablative effect on exophytic tumors is higher because these tumors are easy to target with the rfa probe and because the insulating effect of the surrounding perirenal fat allows the achievement of higher temperatures during rfa. ureteral protection should be considered when laparoscopic rfa is to be performed in a tumor near the renal pelvis and ureter. thus, cases in which the tumors are located within 2 cm of the collecting system and great vessels were not offered laparoscopic rfa. in this study, one patient had incomplete ablation among 41 patients (2.44%). with a mean follow - up of 51 months, no distant metastasis has been observed. the results of this study demonstrate that patients with srms matched for location and size had less ebl, shorter operation time, and shorter hospital stay after laparoscopic rfa than after opn. pettus. reported that a single rfa session for a solitary renal mass did not affect the glomerular filtration rate. nevertheless, this study also concluded that rfa is superior to opn in terms of the patient 's general condition. furthermore, shifts in renal function were not found to be related to tumor size or location but rather to creatinine clearance. recently, another report concluded that rfa achieves moderate local control (5-year disease - free survival, 74%) and may be especially appropriate in elderly patients with a short life expectancy who prefer local treatment. the reported complications include perinephric hematoma, gross hematuria, pyonephrosis, ureteral stricture, damages to adjacent organs, pain, and paresthesias. in addition, rfa of a central tumor can cause other complications including av fistula, segmental infarction, and urinary obstruction [28 - 30 ]. although one patient had an upper ureteral stricture and renal shrinkage after rfa (1/41, 2.44%) and needed to undergo nephrectomy, the case was an early case of our center and did not strictly meet our inclusion criteria for laparoscopic rfa. first, the data were retrospective, introducing the potential for selection bias and additional confounders. second, given factors such as referral patterns to our institution and the wide variability in rfa devices among other institutions, our outcomes may not by generalizable to other centers. our data suggest excellent therapeutic outcomes with laparoscopic rfa with achievement of effective operative times, hospital stays, and ebl compared with opn. there were no significant differences in the complication rates or failure between the two procedures. also, preservation of renal function did not differ significantly between the procedures. according to our operative indication of an exophytic single srm, the indications for laparoscopic rfa were an srm (size <3 cm) for which the collecting system, renal calyx, and great vessel were free from the tumor margins by 1 cm. laparoscopic rfa is an effective minimally invasive therapy for the treatment of srms, yielding long - term oncologic outcomes equivalent to those of opn. prospective randomized studies in diverse patient populations will help to further define the role of laparoscopic rfa as an acceptable treatment alternative to surgery for the definitive management of srms. | purposewe have performed both open partial nephrectomy (opn) and laparoscopic radiofrequency ablation (rfa) on selected patients since january 2007 and have been following these patients through serial laboratory assessments and computed tomography (ct). the purpose of the present study was to evaluate long - term oncologic outcomes and renal function status for laparoscopic rfa versus opn at a minimum follow - up of 3 years.materials and methodsa total of 55 patients with exophytic, single small renal masses were treated with either opn (n=14) or laparoscopic rfa (n=41) by a single surgeon. the indications for laparoscopic rfa were as follows : 1) cases with the greatest dimension of the renal mass <3 cm, and 2) cases in which the collecting system, renal calyx, and great vessels were free from the tumor margins by 1 cm.resultsthe estimated blood loss (ebl), the operation time, and the mean number of hospital days was significantly lower in the laparoscopic rfa group than in the opn group. oncologic data did not differ significantly between the two groups. creatine clearance levels did not differ significantly compared with those before the operation in either group.conclusionsour data suggest excellent therapeutic outcomes with laparoscopic rfa with achievement of effective operative times, hospital stays, and ebl compared with opn. according to our indications for laparoscopic rfa, laparoscopic rfa is an effective minimally invasive therapy for the treatment of small renal masses, yielding oncologic outcomes and renal function equivalent to those of opn. |
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